PERK Inhibition Research Articles

Porcine Circovirus 2 Deploys PERK Pathway and GRP78 for Its Enhanced Replication in PK-15 Cells.

Porcine circovirus type 2 (PCV2) infection induces autophagy and apoptosis. These cellular responses could be connected with endoplasmic reticulum (ER) stress. It remains unknown if PCV2 induces ER stress and if autophagy or apoptosis is primary to PCV2 infection or secondary responses following ER stress. Here, we demonstrate that PCV2 triggered unfolded protein response (UPR) in PK-15 cells by activating the PERK/eIF2α pathway without concomitant activation of IRE1 or ATF6. Since ATF4 and CHOP were induced later than PERK/eIF2α, it is clear that persistent PCV2 infection could lead to selective activation of PERK via the PERK-eIF2α-ATF4-CHOP axis. Therefore, PERK activation could be part of the pro-apoptotic signaling via induced expression of CHOP by PCV2. Since PERK inhibition by GSK2606414 or RNA silencing or suppression of eIF2α dephosphorylation by salubrinal limited viral replication, we suppose that PCV2 deploys UPR to enhance its replication. Over-expression of GRP78 or treatment with tauroursodeoxycholic acid could enhance viral capsid expression and/or viral titers, indicating that these chaperones, endogenous or exogenous, could help correct folding of viral proteins. Our findings provide the first evidence that ER stress plays a role in the pathogenesis of PCV2 infection probably as part of autophagic and apoptotic responses.

Repression of the eIF2α kinase PERK alleviates mGluR-LTD impairments in a mouse model of Alzheimer's disease

Mounting evidence indicates that impairments of synaptic efficacy and/or plasticity may be a key step in the development of Alzheimer's disease (AD) pathophysiology. Among the 2 major forms of synaptic plasticity, long-term potentiation and long-term depression (LTD), much less is known about how LTD is regulated in AD and its molecular mechanisms. Recent studies indicate that metabotropic glutamate receptor 5 (mGluR5) may function as a receptor and/or co-receptor for amyloid beta. Herein, we examined mGluR-LTD in hippocampal slices from aged APP/PS1 mutant mice that model AD. Our findings demonstrate that mGluR-LTD is blocked in APP/PS1 mice, and that the mGluR-LTD failure is reversed by either genetically or pharmacologically suppressing the activity of PERK, a kinase for the mRNA translation factor eIF2α. These data are congruent with recent evidence that inhibition of eIF2α phosphorylation via PERK suppression and reversal of de novo protein synthesis deficits can mitigate cognitive deficits in neurodegenerative diseases. Together with reports indicating that mGluR5 may mediate amyloid beta synaptotoxicity, our findings offer insights into novel therapeutic targets for AD and other cognitive syndromes.

Primary cross-resistance to BRAFV600E-, MEK1/2- and PI3K/mTOR-specific inhibitors in BRAF-mutant melanoma cells counteracted by dual pathway blockade.

Intrinsic cross-resistance to inhibition of different signaling pathways may hamper development of combinatorial treatments in melanoma, but the relative frequency of this phenotype and the strategies to overcome this hurdle remain poorly understood. Among 49 BRAF-mutant melanoma cell lines from patients not previously treated with target therapy, 21 (42.9%) showed strong primary resistance (IC50 > 1 μM) to a BRAFV600E inhibitor. Most of the BRAF-inhibitor-resistant cell lines showed also strong or intermediate cross-resistance to MEK1/2- and to PI3K/mTOR-specific inhibitors. Primary cross-resistance was confirmed in an independent set of 23 BRAF-mutant short-term melanoma cell cultures. MEK1/2 and PI3K/mTOR co-targeting was the most effective approach, compared to BRAF and PI3K/mTOR dual blockade, to counteract primary resistance to BRAF inhibition and the cross-resistant phenotype. This was shown by extensive drug interaction analysis, tumor growth inhibition assays in-vivo, p-ERK and p-AKT inhibition, promotion of melanoma apoptosis, apoptosis-related protein modulation, activation of effector caspases and selective modulation of genes involved in melanoma drug resistance and belonging to the ERK/MAPK and PI3K/AKT canonical pathways. Compared to co-targeting of mutant BRAF and PI3K/mTOR, the association of a MEK1/2 and a PI3K/mTOR inhibitor was more effective in the activation of Bax and of caspase-3 and in the induction of caspase-dependent melanoma apoptosis. Furthermore Bax silencing reduced the latter effects. These results suggest that intrinsic resistance to BRAF inhibition is frequently associated with primary cross-resistance to MEK and PI3K/mTOR blockade in BRAF-mutant melanoma and provide pre-clinical evidence for a combinatorial approach to counteract this phenotype.

EPIG-03EPIGENETIC SUPPRESSION OF EGFR SIGNALING IN G-CIMP + GLIOBLASTOMAS

While it is understood that glioblastomas with the Glioma CpG island methylator phenotype (G-CIMP) phenotype harbor extensive methylation in the CpG islands of a large number of genetic loci, how these methylations patterns translate into the modulation intrinsic signaling cascades remain poorly understood. Using independent, published mRNA signatures associated with EGFR activation, we demonstrate that G-CIMP+ tumors harbor decreased EGFR signaling in three independent datasets, including the REMBRANDT dataset (n = 288), The Cancer Genome Atlas (TCGA; n = 406), and the Chinese Glioma Genome Atlas (CGGA; n = 155). Supporting these findings, analysis of an independent collection of 25 fresh-frozen glioblastomas confirmed lowered pERK levels in G-CIMP+ specimens (p < 0.001), supporting suppressed EGFR signaling in G-CIMP+ glioblastomas. To better understand the mechanism underlying this suppression, we analyzed the TCGA glioblastomas database for the expression level of genes required for EGFR signaling and found that G-CIMP+ glioblastomas harbored lowered mRNA levels for EGFR and H-Ras. In independent glioblastoma models, induction of G-CIMP+ state by exogenous expression of a mutated isocitrate dehydrogenase 1, IDH1-R132H, or by chronic 2-hydroxyglurate treatment suppressed EGFR and H-Ras protein expression as well as pERK accumulation. These suppressions were associated with increased deposition of the repressive histone markers, H3K9me3 and H3K27me3, in the EGFR and H-Ras promoter regions. The IDH1-R132H expression-induced pERK suppression can be reversed by exogenous expression of H-RasG12V but not be exogenous expression of EGFRvIII, suggesting epigenetic suppression H-Ras as the rate limiting step of EGFR signaling in G-CIMP+ glioblastomas. Further supporting suppressed EGFR signaling in G-CIMP+ glioblastomas, we observed increased resistance to the EGFR inhibitor, Gefitnib, in an engineered G-CIMP+ Ink4a-Arf-/- EGFRvIII glioblastoma line relative to its isogenic G-CIMP- counterpart. These results suggest that mitogenic signaling cascades in glioblastomas are epigenetically regulated and a role for IDH1 mutation serves as a predictive biomarker for EGFR inhibitor sensitivity in glioblastoma patients.

PERK inhibition prevents tau-mediated neurodegeneration in a mouse model of frontotemporal dementia.

The PERK-eIF2α branch of the Unfolded Protein Response (UPR) mediates the transient shutdown of translation in response to rising levels of misfolded proteins in the endoplasmic reticulum. PERK and eIF2α activation are increasingly recognised in postmortem analyses of patients with neurodegenerative disorders, including Alzheimer’s disease, the tauopathies and prion disorders. These are all characterised by the accumulation of misfolded disease-specific proteins in the brain in association with specific patterns of neuronal loss, but the role of UPR activation in their pathogenesis is unclear. In prion-diseased mice, overactivation of PERK-P/eIF2α-P signalling results in the sustained reduction in global protein synthesis, leading to synaptic failure, neuronal loss and clinical disease. Critically, restoring vital neuronal protein synthesis rates by inhibiting the PERK-eIF2α pathway, both genetically and pharmacologically, prevents prion neurodegeneration downstream of misfolded prion protein accumulation. Here we show that PERK-eIF2α-mediated translational failure is a key process leading to neuronal loss in a mouse model of frontotemporal dementia, where the misfolded protein is a form of mutant tau. rTg4510 mice, which overexpress the P301L tau mutation, show dysregulated PERK signalling and sustained repression of protein synthesis by 6 months of age, associated with onset of neurodegeneration. Treatment with the PERK inhibitor, GSK2606414, from this time point in mutant tau-expressing mice restores protein synthesis rates, protecting against further neuronal loss, reducing brain atrophy and abrogating the appearance of clinical signs. Further, we show that PERK-eIF2α activation also contributes to the pathological phosphorylation of tau in rTg4510 mice, and that levels of phospho-tau are lowered by PERK inhibitor treatment, providing a second mechanism of protection. The data support UPR-mediated translational failure as a generic pathogenic mechanism in protein-misfolding disorders, including tauopathies, that can be successfully targeted for prevention of neurodegeneration.

Abstract 3272: Targeting LIN28 and the RAS/MAP kinase pathway in atypical teratoid rhabdoid tumors

Abstract Atypical teratoid rhabdoid tumor (AT/RT) is a highly malignant central nervous system neoplasm that primarily affects very young children and has a very poor prognosis. Aside from presumed founder loss of function mutations in the SMARCB1 (BAF47/INI1/SNF5) chromatin remodeling gene, little is known of molecular drivers of AT/RT. LIN28A and LIN28B are stem cell factors that regulate thousands of RNAs and are expressed in aggressive cancers. One of the canonical downstream targets of LIN28 is the RAS/MEK/ERK pathway. Due to the increased stem cell factor expression in AT/RT, we hypothesized that LIN28 contributes to tumorigenesis in these neoplasms through the regulation of multiple pro-growth, stemness, and metabolic pathways. We identified increased levels of LIN28A in AT/RT primary tumors compared to normal brain using immunohistochemistry (P = 0.026 by Mann-Whitney test). We detected LIN28A or LIN28B in 100% of AT/RT cell lines by western blot or qPCR. Knockdown of LIN28A by lentiviral shRNA in the AT/RT cell lines CHLA-06-ATRT and BT37 inhibited growth, cell proliferation and colony formation and induced apoptosis. Colonies formed by BT37 and CHLA-06 cells with LIN28A knockdown were reduced by between 50 and 90 percent (BT37 p = 0.0002 sh800 vs pLKO, CHLA-06 p = 0.009 sh802 vs pLKO). A Cleaved caspase 3 (CC3) assay for apoptosis showed that LIN28A knockdown in BT37 and CHLA-06 cells led to a 4 to 6 fold increase in the percentage of cells expressing CC3 compared to controls measured by immunofluorescence (BT37 p = 0.0005 sh800 vs pLKO; CHLA-06 p = 0.004 sh802 vs pLKO). Suppression of LIN28A in orthotopic xenograft models led to a more than doubling of median survival compared to empty vector controls (48 vs 115 days - p = 0.007 by Log-rank test) showing that LIN28A is critical to AT/RT cell line tumorigenesis. We found lower KRAS expression in LIN28A knockdown cell lines compared to pLKO control using qPCR. We also found high expression levels of phospho-ERK in AT/RT primary tumors by immunohistochemistry. Increased ERK signaling correlated with LIN28A expression in AT/RT (P = 0.008, r = 0.57 by Spearman correlation). Trametinib is a MEK1 and MEK2 inhibitor that has been developed through phase III clinical trials in adult melanoma. Trametinib inhibited the KRAS pathway in CHLA-06-ATRT and BT37 AT/RT cell lines as measured by suppression of p-ERK by Western blot. Treatment with Trametinib for 5 days suppressed the growth of BT37 and CHLA-06 cells (MTS assay for BT37 p = 0.003 for 1 nM and p = 0.002 for 10 nM vs DMSO control; for CHLA-06 p&amp;lt;0.001 for 1 nM and 10nM vs DMSO control by t-test). These data implicate LIN28/RAS/MAP kinase as key drivers of AT/RT tumorigenesis and indicate that targeting this pathway may serve as a novel therapeutic option in this aggressive pediatric malignancy. Citation Format: Jeffrey Rubens, Melanie Weingart, Antoinette Price, Marianne Hutt-Cabezas, Isabella Taylor, Sariah Allen, Brent Orr, Charles Eberhart, Eric Raabe. Targeting LIN28 and the RAS/MAP kinase pathway in atypical teratoid rhabdoid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3272. doi:10.1158/1538-7445.AM2015-3272

Abstract 3323: Radiation-induced ER stress contributes to survival in glioblastoma cell lines

Abstract Glioblastoma is the most common primary malignant brain tumor in adults in the United States. While the standard of care for afflicted patients has evolved to include surgery, ionizing radiation (IR) and temozolomide chemotherapy, the prognoses of these patients remain dismal. With the median survival of glioblastoma patients remaining in the range of 12-15 months, there is an unmet need for approaches that can overcome the inherent therapeutic resistance of these tumors. In recent years, several studies have demonstrated that IR can activate pro-survival signaling that may facilitate adaptation to therapy and the development of radio-resistance. The endoplasmic reticulum stress response (ERSR) is a conserved program known to be deregulated in cancer and can mediate pro-survival signaling in the context of therapy. The ATF6, IRE1 and PERK pathways of the ERSR are known to be important mediators in the initiation of this signaling. In this research, we show for the first time that IR, a key component in glioblastoma therapy, can induce genes downstream of ATF6, IRE1 and PERK in glioblastoma cell lines. Furthermore, we identify the ATF6 and PERK-eIF2a-ATF4 pathways as important contributors to the radiation-response in glioblastoma. Our study began with the observation of protein and mRNA induction of targets associated with the ERSR in irradiated glioblastoma cell lines. In addition to ATF6 target genes, we observed robust induction of PERK target genes 48h after 6 Gy IR. This induction of PERK target genes was accompanied with increased phosphorylation of eIF2a in a time and dose-dependent manner. To evaluate the requirement of PERK for radiation-induced eIF2a phosphorylation, we used a specific inhibitor of PERK - GSK2606414, and found that PERK inhibition was sufficient to prevent radiation-induced eIF2a phosphorylation. We also analyzed expression of several genes downstream of PERK after treatment with GSK2606414, and found that PERK inhibition lead to 70-80% reduction in radiation-induction of downstream genes. Since ATF4 is downstream of PERK-eIF2a, we examined ATF4 expression in irradiated glioblastoma and found that it was also induced by radiation. Using RNA-interference, we found that knockdown of ATF4 reduced proliferation and clonogenic survival by 50%, and resulted in 38% increase in PARP cleavage in irradiated glioblastoma. We observed similar trends when we knocked down ATF6, suggesting that multiple aspects of the ERSR can contribute to the radiation response in glioblastoma. These results indicate that IR-induced activation of ER-stress signaling through PERK and ATF6 contributes to adaptive survival mechanisms in glioblastoma. Further characterization of the mechanism by which ATF6 and ATF4 mediate cell survival after irradiation may reveal novel targets for the enhancement of radiotherapy for glioblastoma. Citation Format: David Dadey, Vaishali Kapoor, Arpine Khudanyan, Dinesh Thotala, Dennis Hallahan. Radiation-induced ER stress contributes to survival in glioblastoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3323. doi:10.1158/1538-7445.AM2015-3323

Abstract LB-244: Amino acid deprivation selectively targets multidrug-resistant breast cancer cells

Abstract Tumor recurrence and metastasis underlie the majority of cancer-related deaths. Cancer cells that recur or metastasize are often both de-differentiated and multidrug resistant, but the mechanistic basis for this has been poorly understood. We have recently shown that de-differentiation promotes multidrug resistance by activating Nrf2, which stimulates transcription of drug efflux pumps and enzymes that scavenge reactive oxygen species (ROS). De-differentiation activates Nrf2 by a non-canonical mechanism involving its phosphorylation by the ER membrane kinase PERK. PERK-Nrf2 signaling protects de-differentiated cells from chemotherapy, and inhibiting this signaling axis re-sensitizes de-differentiated cancer cells to treatment. To further explore this pathway we profiled the effects of PERK inhibition on global gene expression in both differentiated and de-differentiated cells upon treatment with chemotherapy. This analysis showed that PERK inhibition results in an amino acid deprivation phenotype, and suggested that de-differentiated cells may be sensitive to perturbations in amino acid availability. Consistent with this, we found that the aminopeptidase inhibitor Tosedostat was selectively toxic to de-differentiated breast cancer cells when given in combination with chemotherapy. Our findings identify a novel vulnerability of therapy-resistant breast cancer cells, and suggest that targeting amino acid availability in combination with chemotherapy could be an effective treatment for aggressive breast cancers that are multidrug resistant. Citation Format: Catherine A. Del Vecchio, Yuxiong Feng, Ethan S. Sokol, Erik J. Tillman, Sandhya Sanduja, Ferenc Reinhardt, Piyush B. Gupta. Amino acid deprivation selectively targets multidrug-resistant breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-244. doi:10.1158/1538-7445.AM2015-LB-244

Abstract 2677: Decrease in phospho-S6 expression under MEK inhibitor (MEKi) treatment as a potential predictive biomarker of response to MEKi alone or in combination with PI3K-mTOR pathway inhibitor in pancreatic adenocarcinoma in vitro and ex vivo models

Abstract Context: Activating KRAS mutations are frequent (&amp;gt;90%) in PDAC and drive downstream deregulation of both MAPK and PI3K-mTOR pathways. MEK inhibitors (MEKi) are under clinical evaluation in PDAC, in combination with other agents including PI3K-mTOR inhibitors. RAS and BRAF mutations, EMT, PI3K-mTOR activation, and pERK inhibition under treatment have been suggested as predictive markers for MEKi, but remain unvalidated in PDAC. We explored the cellular and molecular effects of MEKi GSK1120212 (GSK212) alone or in combination with PI3K-mTOR inhibitors, in PDAC cell lines and ex vivo culture system. Material and methods: GSK212 is an allosteric non-ATP competitive MEKi. Everolimus (Ev) is a mTORC1 inhibitor and BKM120 a pan-class PI3K inhibitor. Effects on proliferation were evaluated by MTT assay. Combinations were analyzed by the Chou-Talalay method. Protein expression was assessed by Western blot. Ex vivo drug evaluation used selected concentrations of drugs on cultures of fresh tissue slices from patient surgical specimens. Apoptosis and proliferation were evaluated by caspase 3 and MIB-1 (Ki67) immunostaining, respectively. Correlations between protein expressions were explored using linear regression. Results: MIAPaCa-2 and PANC-1 are two mesenchymal KRAS mutated/BRAF wild-type PDAC cell lines with very different response to GSK212, MIAPaCa-2 being sensitive (72h-IC50 = 0.009μM) and PANC-1 resistant (72h-IC50 = 33μM). MIAPaCa-2 was more sensitive than PANC-1 to Ev (IC50 = 23.3μM vs 47.0μM) and BKM120 (IC50 = 4.19μM vs 31.6μM). Combination of GSK212 and Ev or BKM120 for 72h resulted in synergistic effects in MIAPaCa-2 sensitive cell line but not in PANC-1. GSK212 (0.1μM) treatment resulted in pERK extinction in both cell lines but in decreased pS6 expression in MIAPaCa-2 sensitive cell line only. Combination therapy resulted in extinction of pERK and pS6 expression in both cell lines. Apoptosis induction in MIAPaCa-2 was confirmed by PARP cleavage from 48h. Two tumor specimens were cultured ex vivo: (a) GSK212 (0.1 μM) treatment for 48h induced apoptosis (caspase 3 expression) concomitantly with a decrease in pS6 expression; (b) Ev (1μM) or BKM120 (0.1μM) exerted antiproliferative effects but did not induce apoptosis; (c) combinations resulted in higher caspase 3 expression associated with a higher decrease in pS6 expression compared to MEKi alone. Linear regression showed significant correlation between caspase 3 and pS6 (R2 = 0.5798, p = 0.0282, and R2 = 0.9423, p = 0.013, for tumor 1 and 2, respectively). Conclusion: Response to combined MEK/mTOR pathway inhibition was not correlated with “classical” biomarkers of response to MEKi. Decrease in pS6 expression under MEKi treatment may be an early monitoring biomarker of response to MEKi and MEK/mTOR pathway inhibitor combination. Citation Format: Cindy Neuzillet, Annemilaï Tijeras-Raballand, Jérôme Cros, Pierre Bourgoin, Philippe Bourget, Maria Serova, Armand De Gramont, Sandrine Faivre, Eric Raymond, Philippe Ruszniewski, Pascal Hammel. Decrease in phospho-S6 expression under MEK inhibitor (MEKi) treatment as a potential predictive biomarker of response to MEKi alone or in combination with PI3K-mTOR pathway inhibitor in pancreatic adenocarcinoma in vitro and ex vivo models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2677. doi:10.1158/1538-7445.AM2015-2677

Perk inhibition improves structural and functional abnormalities in tau transgenic mice

Perk inhibition improves structural and functional abnormalities in tau transgenic mice

Correlation of decreased pS6 expression under MEK inhibitor (MEKi) with response to MEKi alone or combined with PI3K-mTOR inhibitor (PI3Ki-mTORi) in pancreatic adenocarcinoma (PAC) in vitro and ex vivo models.

e15262 Background: MEKi are under clinical evaluation in combination with other agents including PI3Ki-mTORi. RAS/RAF mutations, EMT, PI3K-mTOR activation, and pERK inhibition during treatment have...

Autophagy and protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2 alpha kinase (eIF2α) pathway protect ovarian cancer cells from metformin-induced apoptosis.

Metformin, an oral biguanide for the treatment of type II diabetes, has been shown to have anticancer effects in ovarian cancer. Energy starvation induced by metformin causes endoplasmic reticulum stress-mediated unfolded protein response (UPR) and autophagy. UPR and autophagy act as a survival or death mechanism in cells. In this study, we observed that metformin-induced apoptosis was relieved by autophagy and the PERK/eIF2α pathway in ovarian cancer cells, but not in peripheral blood mononuclear cells (PBMC) or 'normal' ovarian surface epithelial cells (OSE). Increased PARP cleavage and increased LC3B-II with ATG5-ATG12 complex suggested the induction of apoptosis and autophagy, respectively, in metformin-treated ovarian cancer cells. Accumulation of acidic vacuoles in the cytoplasm and downregulation of p62 further supported late-stage autophagy. Interestingly, metformin induced interdependent activation between autophagy and the UPR, especially the PERK/eIF2α pathway. Inhibition of autophagy-induced PERK inhibition, and vice versa, were demonstrated using small molecular inhibitors (PERK inhibitor I, GSK2606414; autophagy inhibitor, 3-MA, and BafA1). Moreover, autophagy and PERK activation protected ovarian cancer cells against metformin-induced apoptosis. Metformin treatment in the presence of inhibitors of PERK and autophagy, however, had no cytotoxic effects on OSE or PBMC. In conclusion, these results suggest that inhibition of autophagy and PERK can enhance the selective anticancer effects of metformin on ovarian cancer cells. © 2015 Wiley Periodicals, Inc.

Curcumin Modulates Macrophage Polarization Through the Inhibition of the Toll-Like Receptor 4 Expression and its Signaling Pathways

Background: Curcumin, the active ingredient in curcuma rhizomes, has a wide range of therapeutic effects. However, its atheroprotective activity in human acute monocytic leukemia THP-1 cells remains unclear. We investigated the activity and molecular mechanism of action of curcumin in polarized macrophages. Methods: Phorbol myristate acetate (PMA)-treated THP-1 cells were differentiated to macrophages, which were further polarized to M1 cells by lipopolysaccharide (LPS; 1 µg/ml) and interferon (IFN)-γ (20 ng/ml) and treated with varying curcumin concentrations. [³H]thymidine (³H-TdR) incorporation assays were utilized to measure curcumin-induced growth inhibition. The expression of tumor necrosis factor-a (TNF-a), interleukin (IL-6), and IL-12B (p40) were measured by quantitative real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Macrophage polarization and its mechanism were evaluated by flow cytometry and western blot. Additionally, toll-like receptor 4 (TLR4) small interfering RNA and mitogen-activated protein kinase (MAPK) inhibitors were used to further confirm the molecular mechanism of curcumin on macrophage polarization. Results: Curcumin dose-dependently inhibited M1 macrophage polarization and the production of TNF-a, IL-6, and IL-12B (p40). It also decreased TLR4 expression, which regulates M1 macrophage polarization. Furthermore, curcumin significantly inhibited the phosphorylation of ERK, JNK, p38, and nuclear factor (NF)-γB. In contrast, SiTLR4 in combination with p-JNK, p-ERK, and p-p38 inhibition reduced the effect of curcumin on polarization. Conclusions: Curcumin can modulate macrophage polarization through TLR4-mediated signaling pathway inhibition, indicating that its effect on macrophage polarization is related to its anti-inflammatory and atheroprotective effects. Our data suggest that curcumin could be used as a therapeutic agent in atherosclerosis.

495 Divergent androgen regulation of UPR pathways drives prostate cancer

Background: MLN2480 is an orally administered, investigational small molecule pan-RAF kinase inhibitor currently in Phase 1 clinical development. Here, we built integrated PK/PD/efficacy models to understand the relationship between MAPK pathway inhibition and efficacy as well as the effect of dose schedule in two melanoma xenograft models, A375 and SKMEL-2. Materials and Methods: We fit a one-compartment PK model to MLN2480 plasma concentration time profiles following a single dose in mice. Tumor pERK levels in xenograft-bearing mice, measured by Western blotting following single (A375) or multiple (SKMEL-2) doses of MLN2480 were fit to a direct or indirect inhibitory Emax model, respectively, to describe the PK/PD relationship between the plasma concentration and %pERK inhibition. Activity in each xenograft was evaluated as the percent growth rate inhibition (%GRI), the percent change between treated and control exponential tumor volume growth rates over the treatment cycle. To connect PK, PD, and efficacy for each xenograft, we simulated the PK and PD profiles over the treatment cycle for each dose group, and estimated the averages of plasma concentration (Cavg) and %pERK inhibition, respectively, using non-compartmental analysis. Results: From a multiple linear regression, schedule-related parameters are not predictive for drug effect demonstrating schedule-independent activity. For a variety of dose schedules, the relationship between total dose and SKMEL-2 xenograft growth rate is approximately linear (R = 0.90, p < 0.0001). A sigmoidal PK/efficacy model captures the relationship between Cavg and %GRI for both xenografts. A375 exhibits greater sensitivity than SKMEL-2 to the same Cavg of MLN2480, with the models predicting tumor regression (Emax = 172% GRI) and stasis (Emax = 102% GRI) as the maximal effects, respectively. Both xenografts have steep, sigmoidal PD/efficacy relationships, which show the same pERK inhibition is associated with greater %GRI in A375, and %GRI saturates with residual pERK (EC90 = 38% in A375, 22% in SKMEL-2). Conclusions: Integrated PK/PD and PK/efficacy modeling for MLN2480 response in A375 and SKMEL-2 xenografts shows a strong relationship between pERK inhibition and preclinical activity, and that maximal effect is achieved without complete inhibition of the MAPK pathway. The findings of pERK inhibition-dependent but not schedule-dependent activity provide translational guidance to clinical dose and schedule selection.

494 LPA6 promotes growth and tumorigenicity of hepatocellular carcinoma via activation of PIM-3 proto-oncogene kinase

Background: MLN2480 is an orally administered, investigational small molecule pan-RAF kinase inhibitor currently in Phase 1 clinical development. Here, we built integrated PK/PD/efficacy models to understand the relationship between MAPK pathway inhibition and efficacy as well as the effect of dose schedule in two melanoma xenograft models, A375 and SKMEL-2. Materials and Methods: We fit a one-compartment PK model to MLN2480 plasma concentration time profiles following a single dose in mice. Tumor pERK levels in xenograft-bearing mice, measured by Western blotting following single (A375) or multiple (SKMEL-2) doses of MLN2480 were fit to a direct or indirect inhibitory Emax model, respectively, to describe the PK/PD relationship between the plasma concentration and %pERK inhibition. Activity in each xenograft was evaluated as the percent growth rate inhibition (%GRI), the percent change between treated and control exponential tumor volume growth rates over the treatment cycle. To connect PK, PD, and efficacy for each xenograft, we simulated the PK and PD profiles over the treatment cycle for each dose group, and estimated the averages of plasma concentration (Cavg) and %pERK inhibition, respectively, using non-compartmental analysis. Results: From a multiple linear regression, schedule-related parameters are not predictive for drug effect demonstrating schedule-independent activity. For a variety of dose schedules, the relationship between total dose and SKMEL-2 xenograft growth rate is approximately linear (R = 0.90, p < 0.0001). A sigmoidal PK/efficacy model captures the relationship between Cavg and %GRI for both xenografts. A375 exhibits greater sensitivity than SKMEL-2 to the same Cavg of MLN2480, with the models predicting tumor regression (Emax = 172% GRI) and stasis (Emax = 102% GRI) as the maximal effects, respectively. Both xenografts have steep, sigmoidal PD/efficacy relationships, which show the same pERK inhibition is associated with greater %GRI in A375, and %GRI saturates with residual pERK (EC90 = 38% in A375, 22% in SKMEL-2). Conclusions: Integrated PK/PD and PK/efficacy modeling for MLN2480 response in A375 and SKMEL-2 xenografts shows a strong relationship between pERK inhibition and preclinical activity, and that maximal effect is achieved without complete inhibition of the MAPK pathway. The findings of pERK inhibition-dependent but not schedule-dependent activity provide translational guidance to clinical dose and schedule selection.

Dopamine inhibits the function of Gr-1+CD115+ myeloid-derived suppressor cells through D1-like receptors and enhances anti-tumor immunity.

MDSCs accumulate in tumor-bearing animals and cancer patients and are a major factor responsible for cancer-induced immunosuppression that limits effective cancer immunotherapy. Strategies aimed at effectively inhibiting the function of MDSCs are expected to enhance host anti-tumor immunity and improve cancer immunotherapy significantly. The neurotransmitter DA has been found to have anti-cancer activity, but the underlying mechanism is poorly understood. In this study, we sought to investigate the therapeutic mechanism and efficacy of DA on the inhibition of cancer development via the regulation of MDSC functions. The regulation of the suppressive function of Gr-1(+)CD115(+) MDSCs by DA was determined by use of murine syngeneic LLC and B16 graft models treated with DA in vivo, as well as Gr-1(+)CD115(+) MDSCs isolated from these model treated with DA ex vivo. Here, we show that Gr-1(+)CD115(+) monocytic MDSCs express D1-like DA receptors. DA dramatically attenuated the inhibitory function of tumor-induced monocytic MDSCs on T cell proliferation and IFN-γ production via D1-like DA receptors and retarded tumor growth. DA and other D1 receptor agonists inhibited IFN-γ-induced NO production by MDSCs from tumor-bearing mice and cancer patients. Decreased NO production was, in part, mediated via the suppression of p-ERK and p-JNK. In conclusion, the neurotransmitter DA potently inhibits the suppressive function of MDSC and enhances anti-tumor immunity. Our finding provides a mechanistic basis for the use of DA or D1-like receptor agonists to overcome tumor-induced immunosuppression in cancer immunotherapy.

Abstract SY44-02: Insights into pro-survival signaling by PERK

Abstract PERK, one of three key signal transducers localized to the Endoplasmic Reticulum (ER), regulates cell fate in response to proteotoxic stress. PERK activation can be triggered by exposure of cells to physiological stresses such as low energy (glucose deprivation), low oxygen (hypoxia), or by chemical that inhibit protein degradation (bortezomib) or deplete ER calcium stores (thapsigargin). Since tumor survival and progression requires bypass of many of the same stresses that are known to activate PERK, extensive efforts have been undertaken to ascertain whether targeted inhibition of PERK would have therapeutic benefit. Indeed, genetic abrogation of PERK signaling has a profound influence on tumor progression and metastasis supporting the development of small molecule PERK inhibitors. Importantly, the successful use of such small molecules in a clinical setting requires knowledge of both the potential tissue toxicities associated with acute PERK inhibition and a detailed understanding of downstream PERK effectors that contribute to cell growth and survival. To definitively establish the functional role of PERK in the adult tissue, we have generated mice harboring a conditional PERK allele where excision is regulated by tamoxifen administration. In contrast to previous reports, PERK deletion has a profound impact on pancreata regardless of age. This result has profound implications for the utility of small molecule PERK inhibitors and highlights the need to clearly delineate downstream PERK-dependent regulatory pathways. With this in mind, we have set out to define additional pro-survival PERK effector and effector pathways in an effort to develop a more complete understanding of PERK function and ascertain whether any novel effectors might represent opportunities for therapeutic development. The identification and contribution of novel PERK effectors will be discussed. Citation Format: J. Alan Diehl. Insights into pro-survival signaling by PERK. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY44-02. doi:10.1158/1538-7445.AM2014-SY44-02

Vemurafenib and Cobimetinib Potently Inhibit Ps6 Signaling in Brafv600 Mutation–Positive Locally Advanced or Metastatic Melanoma from Brim7 Study

ABSTRACT Aim: Combined BRAF and MEK inhibition, compared with BRAF inhibitor alone, may delay the onset of resistance. Vemurafenib + cobimetinib (vem + cobi) was evaluated in patients naive to or previously treated with BRAF inhibitor (BRAFi) in a phase Ib trial: BRIM7 (NCT01271803). Modulation of signaling pathways and transcriptional profiles were assessed in tumor samples. Methods: Tumor tissues were collected at baseline, on-treatment at cycle 1 day 14 (C1D14), and at disease progression (PD). Modulation of cell-signaling pathways was assessed in tumor biopsy specimens by immunohistochemistry (IHC) or reverse-phase protein array (RPPA). Tumor transcriptional profiles were measured on a NanoString platform. Results: pERK levels were inhibited an average of 86 ± 14% with vem + cobi in paired biopsy specimens (n = 7) at C1D14 compared with baseline, which coincided with enhanced down-regulation of ERK transcriptional targets, compared with vem alone (n = 20). Cytoplasmic pERK was inhibited by 92 ± 3% in BRAFi-naive patients and by 95 ± 3% in patients with PR/CR responses. The inhibition of S6 ribosomal protein phosphorylation (pS6) showed significant differences based on treatment response and prior treatment status. By IHC, pS6 levels were clearly reduced in BRAFi-naive patients (82 ± 8%) compared with patients whose disease progressed with BRAFi treatment (31 ± 22%) and similarly in PR/CR patients (88 ± 11%) vs PD/SD patients (33 ± 19%). These observations were verified in independent samples by RPPA: levels of pS6 (S235/236 and S240/244) and a pS6 downstream effector, p4EBP1 (T37/46), were significantly inhibited in the same patients. At PD, tumor pERK, pS6, and Ki67 inhibition were attenuated to different degrees in each patient, whereas transcriptional programs largely reverted to baseline profiles. Conclusions: Vem + cobi treatment resulted in robust modulation of tumor cell signaling and transcriptional programs. In concordance with reports for single-agent BRAF or MEK inhibitor treatment, potent inhibition of pS6 was observed in BRAFi-naive patients and patients with PR/CR, which may contribute to the enhanced response to the vem + cobi combination. Disclosure: Y. Yan: Employee of Genentech, Inc.; G. McArthur: Consultant: Roche-Genentech, Novartis, GSK, BMS, Millenium, Amgen Research: Pfizer, Millenium, Novartis; T. Gajewski: Consultant: Bayer, B-I, Abbvie, Roche/Genentech, Jounce, Dendreon, Amgen Investigator: BMS, Merck, Roche/Genentech, Incyte, Ono; I. Puzanov: Consultant: Roche (honoraria) Investigator: Roche/Genentech (funds to university); O. Hamid: Research/advisory board/consultant/speaker: Genentech; R. Gonzalez: Research/consultant/honoraria: GSK, Roche/Genentech, BMS; Y. Wang: Employee: Genentech; S. Lu: Employee: Genentech; M. Wongchenko: Employee: Genentech; N.W. Choong: Employee: Roche/Genentech Stockholder: Roche; A. Ribas: Consultant: Amgen, GSK, Genentech, Merck Stockholder: Kite Pharma.

Epigenetic suppression of EGFR signaling in G-CIMP+ glioblastomas.

The intrinsic signaling cascades and cell states associated with the Glioma CpG Island Methylator Phenotype (G-CIMP) remain poorly understood. Using published mRNA signatures associated with EGFR activation, we demonstrate that G-CIMP+ tumors harbor decreased EGFR signaling using three independent datasets, including the Chinese Glioma Genome Atlas(CGGA; n=155), the REMBRANDT dataset (n=288), and The Cancer Genome Atlas (TCGA; n=406). Additionally, an independent collection of 25 fresh-frozen glioblastomas confirmed lowered pERK levels in G-CIMP+ specimens (p<0.001), indicating suppressed EGFR signaling. Analysis of TCGA glioblastomas revealed that G-CIMP+ glioblastomas harbored lowered mRNA levels for EGFR and H-Ras. Induction of G-CIMP+ state by exogenous expression of a mutated isocitrate dehydrogenase 1, IDH1-R132H, suppressed EGFR and H-Ras protein expression as well as pERK accumulation in independent glioblastoma models. These suppressions were associated with increased deposition of the repressive histone markers, H3K9me3 and H3K27me3, in the EGFR and H-Ras promoter regions. The IDH1-R132H expression-induced pERK suppression can be reversed by exogenous expression of H-RasG12V. Finally, the G-CIMP+ Ink4a-Arf-/- EGFRvIII glioblastoma line was more resistant to the EGFR inhibitor, Gefitinib, relative to its isogenic G-CIMP- counterpart. These results suggest that G-CIMP epigenetically regulates EGFR signaling and serves as a predictive biomarker for EGFR inhibitors in glioblastoma patients.

Abstract 659: PKR-Like Endoplasmic Reticulum Kinase and Glycogen Synthase Kinase-3α/ß Signaling Regulates Endoplasmic Reticulum Stress-Induced Foam Cell Formation

Background: Evidence suggests a causative role for endoplasmic reticulum (ER) stress in the development of atherosclerosis. The molecular mechanisms by which conditions of ER stress promote pro-atherogenic processes are not understood. We have found that ER stress-inducing agents can activate glycogen synthase kinase (GSK)-3α/β, a protein involved in many metabolic pathways. The objective of this study is to investigate the role of GSK3α/β in pro-atherogenic ER stress signaling. Methods and Results: Thp1-derived macrophages were treated with the ER stress-inducing agents, glucosamine, thapsigargin or palmitate, in the presence or absence of the GSK3α/β inhibitor CT99021. GSK3α/β inhibition did not affect the adaptive unfolded protein response (UPR), but did block ER stress-induced lipid accumulation as well as the up regulation of genes associated with lipid biosynthesis and uptake. Using small molecule inhibitors of specific UPR pathways, we found that PERK, but not IRE1 or ATF6, is required for the activation of GSK3α/β by ER stress. GSK3α/β inhibition attenuated ER stress-induced expression of distal components of the PERK pathway, including CHOP and ATF4. Atherosclerotic plaques from ApoE-/- mice, fed a diet supplemented with the GSK3α/β inhibitor valproate, had reduced levels of CHOP within the macrophage foam cells. In primary mouse macrophages PERK inhibition blocked ER stress induced lipid accumulation whereas the overexpression of constitutively active S9A-GSK3β promoted foam cell formation and CHOP expression, even in cells treated with a PERK inhibitor. Conclusions: Pharmacological inhibition of GSK3α/β attenuates ER stress-induced lipid accumulation and macrophage foam cell formation. These findings indicate that GSK3α/β is an important factor in the ER stress-PERK signaling pathway and may play a central role in the pro-atherogenic dysregulation of lipid metabolism.