Response Pathway Activation Research Articles

High throughput adaptive stress response pathway activation to support read across of valproic acid analogue-induced liver steatosis

High throughput adaptive stress response pathway activation to support read across of valproic acid analogue-induced liver steatosis

Abstract 765: Multiple signalling pathways are active in BRAF/NRAS wild type melanomas

Abstract In the last five years, the treatment of advanced melanoma has been revolutionised by the introduction of selective kinase inhibitors and immunotherapy. Selective inhibition of the mitogen activated protein kinase (MAPK) pathway has significantly improved the survival of patients with BRAFV600-mutant melanoma, and combination MEK and CDK4 inhibition is currently being trialed in patients with NRAS-mutant melanoma. Almost a quarter of all melanomas, however, are wild type for BRAF and NRAS and have no known actionable mutations. Currently, patients with wild type melanoma are treated with immunotherapies, which produce prolonged responses, but only in a small subset of patients. Overall, the prognosis for patients with wild type melanomas remains poor, and there is an urgent need to establish better therapeutic strategies for these patients. In order to define new therapeutic targets in BRAF/NRAS wild type melanoma, we sought to characterize the key proliferative and survival pathways in wild type melanomas. Wild type (n=14) and BRAFV600 (n=7) melanomas were treated with the MEK inhibitor trametinib at increasing doses from 0.5nM to 5000nM for 72h and sensitivity to MEK inhibition was assessed using viability assays. Signaling pathway activity in response to trametinib (10nM, 24h) was also examined in our panel of melanoma cells using the Human Phospho-Kinase Antibody Array (R&D Systems), which detects 43 different phosphorylated kinases. As expected, BRAFV600 melanomas were exquisitely sensitive to trametinib treatment (IC50=0.8-2.4nM). We also found that although a subset of BRAF/NRAS wild type melanomas (5/14; 36%) were sensitive to trametinib (IC50 0.5-4.9nM), the majority of these melanoma cells (9/14; 64%) were resistant to trametinib (IC50 10.5-264.8nM). Analysis of kinase activation status revealed little difference in signaling pathway activity between the sensitive and resistant wild type melanomas at baseline. Resistant wild type melanomas had higher activity of STAT3 compared to the sensitive cell lines. However, all these wild type melanomas showed variable activation of multiple signaling pathways that were not active in BRAFV600 melanomas. Specifically, at baseline, wild type melanomas showed pronounced activation of the JNK and p38 MAPK pathways, the FAK/Src pathway (i.e. FAK, Lck, Lyn, Fgr and Yes), the epidermal growth factor receptor tyrosine kinase and β-catenin compared to BRAFV600 melanomas. Activation of multiple signaling pathways in wild type melanomas may contribute to innate resistance or limit the efficacy of MEK inhibition, and we are currently exploring the impact of these pathways on the survival and MEK dependence of wild type melanomas. Citation Format: Zizhen Ming, Su Yin Lim, Richard F. Kefford, Helen Rizos. Multiple signalling pathways are active in BRAF/NRAS wild type melanomas [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 765.

Nucleic acid sensing in the cytoplasm of C2C12 cells directly after plasmid DNA electroporation

Abstract Mounting evidence stresses the importance of nucleic acid sensing in the cytoplasm of living cells in a variety of physiological and pathological processes, including anti-pathogen defense, autoimmunity and development of malignant tumors. A subset of pattern-recognition receptors (PRRs) are nucleic acid sensors widely expressed in a variety of cells, but the detailed mechanisms of nucleic acid sensing have not been clarified. Our previous studies confirmed the cell type-specific upregulation of PRRs on both the mRNA and protein levels after electroporation of plasmid DNA (pDNA) into the cells. Type I interferon was found to be upregulated as well, which implicates innate immunity pathway activation in response to pDNA entry into the cytoplasm. Still, the sequence of events after pDNA electroporation remains unclear. Using molecular and proteomic techniques, we identified DAI/ZBP1 protein as the initial responding PRR to pDNA entering the cytoplasm after electroporation and estimated the participation of other PRRs in this process in mouse myoblast C2C12 cells. Better understanding of molecular determinants of nucleic acid sensing by PRRs may allow us to manipulate this process, developing new and improving existing therapies for immunity- and tumor-related diseases.

Dynamic imaging of adaptive stress response pathway activation for prediction of drug induced liver injury

Drug-induced liver injury remains a concern during drug treatment and development. There is an urgent need for improved mechanistic understanding and prediction of DILI liabilities using in vitro approaches. We have established and characterized a panel of liver cell models containing mechanism-based fluorescent protein toxicity pathway reporters to quantitatively assess the dynamics of cellular stress response pathway activation at the single cell level using automated live cell imaging. We have systematically evaluated the application of four key adaptive stress pathway reporters for the prediction of DILI liability: SRXN1-GFP (oxidative stress), CHOP-GFP (ER stress/UPR response), p21 (p53-mediated DNA damage-related response) and ICAM1 (NF-κB-mediated inflammatory signaling). 118 FDA-labeled drugs in five human exposure relevant concentrations were evaluated for reporter activation using live cell confocal imaging. Quantitative data analysis revealed activation of single or multiple reporters by most drugs in a concentration and time dependent manner. Hierarchical clustering of time course dynamics and refined single cell analysis allowed the allusion of key events in DILI liability. Concentration response modeling was performed to calculate benchmark concentrations (BMCs). Extracted temporal dynamic parameters and BMCs were used to assess the predictive power of sub-lethal adaptive stress pathway activation. Although cellular adaptive responses were activated by non-DILI and severe-DILI compounds alike, dynamic behavior and lower BMCs of pathway activation were sufficiently distinct between these compound classes. The high-level detailed temporal- and concentration-dependent evaluation of the dynamics of adaptive stress pathway activation adds to the overall understanding and prediction of drug-induced liver liabilities.

Molecular and clinical characterization of IDH associated immune signature in lower-grade gliomas

ABSTRACTBackground: Mutations in isocitrate dehydrogenase (IDH) affect the development and prognosis of gliomas. We investigated the role of IDH mutations in the regulation of immune phenotype in lower-grade gliomas (LGGs).Method and patients: A total of 1,008 cases with clinical and IDH mutation data from five cohorts were enrolled. Samples with RNA sequencing data from the Chinese Glioma Genome Atlas (CGGA) were used as training set, whereas RNA data from the Cancer Genome Atlas, Repository for Molecular Brain Neoplasia, GSE16011, and CGGA microarray databases were used for validation. R language tools and bioinformatics analysis were used for gene signature construction and biological function annotation.Results: We found that IDH mutations caused down-regulation of local immune response as among 332 immune system-related genes, 196(59.0%) were differentially expressed according to IDH mutation status. Nearly 70% of those differentially expressed genes exhibited prognostic value in LGGs. An immune response-based gene signature was constructed that distinguished cases with high- or low-risk of unfavorable prognosis and remained an independent prognostic factor in multivariate analyses in both training and validation cohorts. Samples from high-risk cases exhibited elevated expression of genes involved in immune response and NF-κB pathway activation. Furthermore, we found a strong correlation between the risk score and T cells, macrophage-related immune response, and expression of several prominent immune checkpoints.Conclusion: Our results indicated that mutant IDH is highly associated with the regulation of the immune microenvironment in LGGs. The observed immune system gene signature, which was sensitive to IDH mutation status, efficiently predicted patient survival.

CO-releasing molecules-2 attenuates ox-LDL-induced injury in HUVECs by ameliorating mitochondrial function and inhibiting Wnt/β-catenin pathway

Oxidized low-density lipoprotein (ox-LDL) is well known to disrupt normal functionality of endothelium, which plays a prominent role in endothelial dysfunction in many cardiovascular diseases. CO-releasing molecule 2 (CORM-2) is a promising candidate for treatment of cardiovascular diseases. However, it has not been defined whether CORM-2 might improve endothelial injury induced by ox-LDL. The present study was undertaken to determine the regulatory role of CORM-2 in cell injury of ox-LDL-treated human umbilical vein endothelial cells (HUVECs). Our results showed that ox-LDL inhibited the cell proliferation, but promoted apoptosis and release of cytochrome c (cytc) from mitochondrion into cytoplasm, stimulated the cleavage of caspase-3 and mitochondrial permeability transition pore (MPTP) opening. In addition, ox-LDL-incubated HUVECs exhibited excessive reactive oxygen species (ROS), increased protein levels of NADPH oxidase subunits p22phox, p47phox, NOX-2 and activation of Wnt/β-catenin signaling pathway. However, pretreatment with CORM-2 significantly reduced cell apoptosis, release of cytc from mitochondrion into cytoplasm, MPTP opening and cleavage of caspase-3, suppressed the superoxide anion generation and Wnt/β-catenin pathway activation in HUVECs response to ox-LDL. Collectively, we provide the evidence that CORM-2 attenuated ox-LDL-mediated endothelial apoptosis and oxidative stress by recovering the mitochondrial function and blocking Wnt/β-catenin pathway.

Type I interferon-regulated gene expression and signaling in murine mixed glial cells lacking signal transducers and activators of transcription 1 or 2 or interferon regulatory factor 9

Type I interferons (IFN-I) are critical in antimicrobial and antitumor defense. Although IFN-I signal via the interferon-stimulated gene factor 3 (ISGF3) complex consisting of STAT1, STAT2, and IRF9, IFN-I can mediate significant biological effects via ISGF3-independent pathways. For example, the absence of STAT1, STAT2, or IRF9 exacerbates neurological disease in transgenic mice with CNS production of IFN-I. Here we determined the role of IFN-I-driven, ISGF3-independent signaling in regulating global gene expression in STAT1-, STAT2-, or IRF9-deficient murine mixed glial cell cultures (MGCs). Compared with WT, the expression of IFN-α-stimulated genes (ISGs) was reduced in number and magnitude in MGCs that lacked STAT1, STAT2, or IRF9. There were significantly fewer ISGs in the absence of STAT1 or STAT2 versus in the absence of IRF9. The majority of ISGs regulated in the STAT1-, STAT2-, or IRF9-deficient MGCs individually were shared with WT. However, only a minor number of ISGs were common to WT and STAT1-, STAT2-, and IRF9-deficient MGCs. Whereas signal pathway activation in response to IFN-α was rapid and transient in WT MGCs, this was delayed and prolonged and correlated with increased numbers of ISGs expressed at 12 h versus 4 h of IFN-α exposure in all three IFN-I signaling-deficient MGCs. In conclusion, 1) IFN-I can mediate ISG expression in MGCs via ISGF3-independent signaling pathways but with reduced efficiency, with delayed and prolonged kinetics, and is more dependent on STAT1 and STAT2 than IRF9; and 2) signaling pathways not involving STAT1, STAT2, or IRF9 play a minor role only in mediating ISG expression in MGCs.

JAK-STAT pathway activation in response to spinal cord injury in regenerative and non-regenerative stages of Xenopus laevis.

Xenopus laevis tadpoles can regenerate the spinal cord after injury but this capability is lost during metamorphosis. Comparative studies between pre‐metamorphic and metamorphic Xenopus stages can aid towards understanding the molecular mechanisms of spinal cord regeneration. Analysis of a previous transcriptome‐wide study suggests that, in response to injury, the JAK‐STAT pathway is differentially activated in regenerative and non‐regenerative stages. We characterized the activation of the JAK‐STAT pathway and found that regenerative tadpoles have an early and transient activation. In contrast, the non‐regenerative stages have a delayed and sustained activation of the pathway. We found that STAT3 is activated in response to injury mainly in Sox2/3+ ependymal cells, motoneurons and sensory neurons. Finally, to study the role of temporal activation we generated a transgenic line to express a constitutively active version of STAT3. The sustained activation of the JAK‐STAT pathway in regenerative tadpoles reduced the expression of pro‐neurogenic genes normally upregulated in response to spinal cord injury, suggesting that activation of the JAK‐STAT pathway modulates the fate of neural progenitors.

Protective effect of quercetin on high-fat diet-induced non-alcoholic fatty liver disease in mice is mediated by modulating intestinal microbiota imbalance and related gut-liver axis activation

Gut microbiota is involved in obesity, metabolic syndrome and the progression of nonalcoholic fatty liver disease (NAFLD). It has been recently suggested that the flavonoid quercetin may have the ability to modulate the intestinal microbiota composition, suggesting a prebiotic capacity which highlights a great therapeutic potential in NAFLD. The present study aims to investigate benefits of experimental treatment with quercetin on gut microbial balance and related gut-liver axis activation in a nutritional animal model of NAFLD associated to obesity. C57BL/6J mice were challenged with high fat diet (HFD) supplemented or not with quercetin for 16 weeks. HFD induced obesity, metabolic syndrome and the development of hepatic steatosis as main hepatic histological finding. Increased accumulation of intrahepatic lipids was associated with altered gene expression related to lipid metabolism, as a result of deregulation of their major modulators. Quercetin supplementation decreased insulin resistance and NAFLD activity score, by reducing the intrahepatic lipid accumulation through its ability to modulate lipid metabolism gene expression, cytochrome P450 2E1 (CYP2E1)-dependent lipoperoxidation and related lipotoxicity. Microbiota composition was determined via 16S ribosomal RNA Illumina next-generation sequencing. Metagenomic studies revealed HFD-dependent differences at phylum, class and genus levels leading to dysbiosis, characterized by an increase in Firmicutes/Bacteroidetes ratio and in Gram-negative bacteria, and a dramatically increased detection of Helicobacter genus. Dysbiosis was accompanied by endotoxemia, intestinal barrier dysfunction and gut-liver axis alteration and subsequent inflammatory gene overexpression. Dysbiosis-mediated toll-like receptor 4 (TLR-4)-NF-κB signaling pathway activation was associated with inflammasome initiation response and reticulum stress pathway induction. Quercetin reverted gut microbiota imbalance and related endotoxemia-mediated TLR-4 pathway induction, with subsequent inhibition of inflammasome response and reticulum stress pathway activation, leading to the blockage of lipid metabolism gene expression deregulation. Our results support the suitability of quercetin as a therapeutic approach for obesity-associated NAFLD via its anti-inflammatory, antioxidant and prebiotic integrative response.

Control of inflammation by stromal Hedgehog pathway activation restrains colitis

Inflammation disrupts tissue architecture and function, thereby contributing to the pathogenesis of diverse diseases; the signals that promote or restrict tissue inflammation thus represent potential targets for therapeutic intervention. Here, we report that genetic or pharmacologic Hedgehog pathway inhibition intensifies colon inflammation (colitis) in mice. Conversely, genetic augmentation of Hedgehog response and systemic small-molecule Hedgehog pathway activation potently ameliorate colitis and restrain initiation and progression of colitis-induced adenocarcinoma. Within the colon, the Hedgehog protein signal does not act directly on the epithelium itself, but on underlying stromal cells to induce expression of IL-10, an immune-modulatory cytokine long known to suppress inflammatory intestinal damage. IL-10 function is required for the full protective effect of small-molecule Hedgehog pathway activation in colitis; this pharmacologic augmentation of Hedgehog pathway activity and stromal IL-10 expression are associated with increased presence of CD4+Foxp3+ regulatory T cells. We thus identify stromal cells as cellular coordinators of colon inflammation and suggest their pharmacologic manipulation as a potential means to treat colitis.

Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory infections that can be life-threatening. To establish an infection and spread, MERS-CoV, like most other viruses, must navigate through an intricate network of antiviral host responses. Besides the well-known type I interferon (IFN-α/β) response, the protein kinase R (PKR)-mediated stress response is being recognized as an important innate response pathway. Upon detecting viral dsRNA, PKR phosphorylates eIF2α, leading to the inhibition of cellular and viral translation and the formation of stress granules (SGs), which are increasingly recognized as platforms for antiviral signaling pathways. It is unknown whether cellular infection by MERS-CoV activates the stress response pathway or whether the virus has evolved strategies to suppress this infection-limiting pathway. Here, we show that cellular infection with MERS-CoV does not lead to the formation of SGs. By transiently expressing the MERS-CoV accessory proteins individually, we identified a role of protein 4a (p4a) in preventing activation of the stress response pathway. Expression of MERS-CoV p4a impeded dsRNA-mediated PKR activation, thereby rescuing translation inhibition and preventing SG formation. In contrast, p4a failed to suppress stress response pathway activation that is independent of PKR and dsRNA. MERS-CoV p4a is a dsRNA binding protein. Mutation of the dsRNA binding motif in p4a disrupted its PKR antagonistic activity. By inserting p4a in a picornavirus lacking its natural PKR antagonist, we showed that p4a exerts PKR antagonistic activity also under infection conditions. However, a recombinant MERS-CoV deficient in p4a expression still suppressed SG formation, indicating the expression of at least one other stress response antagonist. This virus also suppressed the dsRNA-independent stress response pathway. Thus, MERS-CoV interferes with antiviral stress responses using at least two different mechanisms, with p4a suppressing the PKR-dependent stress response pathway, probably by sequestering dsRNA. MERS-CoV p4a represents the first coronavirus stress response antagonist described.

Abstract 2138: Functional evaluation of interferon/STAT1 pathway activation in response to genotoxic treatment

Abstract Triple negative breast cancer (TNBC) accounts for approximately 15-25% of breast cancers at diagnosis, and is one of the most aggressive subtypes, with around 70% of patients that live free of disease 5 years post-diagnosis. One of the most reliable predictive markers of patient outcome is the pathological complete response (pCR), with no tumor cells detectable at histopathological level after neoadjuvant chemotherapy. As pCR is observed in only about 20-30% of TNBC, it is mandatory to identify new therapeutic options to improve pCR rate upon neoadjuvant chemotherapy. The mechanisms of tumor resistance remain an unmet need in oncology. In a recent study, we used our breast cancer PDXs collection to tackle this question, and address the mechanisms of tumor response to treatment vs tumor recurrence. To do this we analyzed the gene expression profile of laser-microdissected residual tumor nodules interspersed in the murine stroma upon very efficient tumor response to Adriamycin/Cyclophosphamide (AC). When doing so, we identified several genes of the IFN/STAT1 pathway that were strongly over-expressed when compared to pre-treatment tumors. In this study, we investigated the mechanisms leading to IFN/STAT1 pathway activation following chemotherapy in tumor cells and we evaluated the functional involvement of the IFN/STAT1 signature as a whole or at single target gene level in residual tumor cell resistance to chemotherapy after genotoxic treatment. To this aim, we set up an ex-vivo/in vitro assay to mimic induction of the IFN/STAT1 signature in tumor cells after genotoxic treatment. We identified cell lines and primary cultures from a patient-derived xenograft (PDX) that mimicked the in vivo situation, as only the cell lines that respond to genotoxic stress in vivo activated IFN/STAT1 pathway in response to genotoxic treatment in vitro. When exposed to conditioned medium collected from mafosfamide-treated cancer cells, these cells activated luciferase reporter genes harboring ISRE (interferon stimulated response elements) and GAS (gamma interferon activated sequence) response elements, suggesting active ligands of the IFN/STAT1 pathways were secreted. STAT1 or IFNAR1 gene silencing (siRNA) resulted in markedly attenuated gene signature expression after mafosfamide treatment. The addition of conditioned medium significantly reduced mafosfamide-induced cancer cell death suggesting that IFN/STAT1-related genes over-expression may ultimately have protective effect on cancer cell viability. Accordingly, the inhibition of several genes of the IFN/STAT pathway, or DNA sensors acting upstream, increased cell mortality when combined with genotoxic treatment and delayed colony formation. Overall, this study supports the functional implication of IFN/STAT1-related genes in tumor resistance to genotoxic treatment. Citation Format: Julie Gaston, Laura Cheradame, Marie-Emmanuelle Legrier, Olivier Deas, Jean Gabriel Judde, Vincent Goffin, Stefano Cairo. Functional evaluation of interferon/STAT1 pathway activation in response to genotoxic treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2138.

Abstract 230: The role of integrated stress response (ISR) transcription factor ATF4 in metabolic adaptation of tumors to c-Myc activation

Abstract The ability of cancer cells to adapt to non-cell autonomous (extrinsic) and cell autonomous (intrinsic) stresses is critical for maintaining cell viability and growth. The Integrated Stress Response (ISR) pathway is essential in cancer cell adaptation to extrinsic stresses such as hypoxia and nutrient deprivation both in vitro and in vivo. The ISR kinases, PERK and GCN2, phosphorylate eIF2α, transiently inhibiting protein translation during times of stress. Phosphorylation of eIF2α promotes the preferential translation of the transcription factor ATF4, which activates a gene expression program that enhances uptake and synthesis of amino acids, antioxidant defense and chaperone expression to promote recovery from nutrient deprivation and ER stress. Here, we demonstrate the importance of ISR signaling in MYC oncogene induced intrinsic stress both in vitro and in vivo. We previously reported that activation of PERK, during c-Myc induction is vital for activating cytoprotective autophagy and suppressing c-Myc induced apoptosis. Here, we demonstrate GCN2 is also robustly activated during c-Myc induction. GCN2 activation after c-Myc induction regulates ATF4 transcription and translation. ATF4 was necessary for survival and suppression of oxidative stress during c-Myc activation. We have identified ATF4 targets that regulate amino acid uptake and involved in amelioration of oxidative stress. Furthermore, siRNA mediated knockdown or genetic knockout of GCN2 was synthetic lethal with c-Myc activation. Interestingly, PERK is still able to phosphorylate eIF2α in the absence of GCN2. However, eIF2α phosphorylation was completely inhibited in the absence of both kinases suggesting that these kinases compensate for each other and both are required for optimal phosphorylation of eIF2α after c-Myc activation. Consistent with our in vitro data, we observe higher levels of p-eIF2α and ATF4 in lymphoma cells compared to WT B cells in vivo in a mouse model of Myc driven lymphoma. These findings highlight a novel mechanism of stress response pathway activation to support c-Myc driven tumorigenesis involving an amino acid sensor kinase GCN2. Citation Format: Feven Tameire, Maria A. Monroy, Constantinos Koumenis. The role of integrated stress response (ISR) transcription factor ATF4 in metabolic adaptation of tumors to c-Myc activation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 230.

TREX1 Knockdown Induces an Interferon Response to HIV that Delays Viral Infection in Humanized Mice.

Despite their antiviral effect, the invivo effect of interferons on HIV transmission is difficult to predict, because interferons also activate and recruit HIV-susceptible cells to sites of infection. HIV does not normally induce type I interferons in infected cells, but does if TREX1 is knocked down. Here, we investigated the effect of topical TREX1 knockdown and local interferon production on HIV transmission in human cervicovaginal explants and humanized mice. Inexplants in which TREX1 was knocked down, HIV induced interferons, which blocked infection. In humanized mice, even though TREX1 knockdown increased infiltrating immune cells, it delayed viral replication for 3-4weeks. Similarly intravaginal application of type I interferons the day before HIV infection induced interferon responsive genes, reduced inflammation, and decreased viral replication. However, intravenous interferon enhanced inflammation and infection. Thus, in models of human sexual transmission, a localized interferon response inhibits HIV transmission but systemic interferons do not.

Identification and functional characterization of a glucose regulated protein 94 gene in Litopenaeus vannamei and its responsiveness in WSSV infection.

In the current study, a cDNA of glucose regulated protein 94 (LvGRP94) was cloned from Litopenaeus vannamei. Subcellular localization assay revealed that LvGRP94 expressed in endoplasmic reticulum (ER). And results of reported gene assays demonstrated that the promoter of LvGRP94 was activated by L. vannamei leucine zipper domain transcription factor X-box binding protein 1 (LvXBP1) or heat shock treatment. Furthermore, LvGRP94 was found to highly express in hemocytes as well as in epidermis by real-time RT-PCR. In addition, it was shown that LvGRP94 inhibited by LvXBP1 knocked-down in the hemocytes, was induced by white spot syndrome virus (WSSV) infection, or unfolded protein response (UPR) pathway activation. Importantly, decreasing LvGRP94 reduced the cumulative mortality of WSSV-infected shrimps and WSSV copies in shrimp muscle. These results suggested that LvGRP94 might involve in shrimp UPR pathway as well as WSSV infection.

Abstract PL03-03: K-RAS dependency in pancreatic cancer

Abstract 2015 AACR-NCI-EORTC K-RAS dependency in Pancreatic Cancer David Tuveson, M.D., Ph.D., Cold Spring Harbor Laboratory Efforts to inhibit the KRAS oncogene in pancreatic cancer have focused on classical downstream pathways and empirically identified dependencies. We have developed organoid models of mouse and human pancreatic cancer to expedite such translational approaches for this disease. We find that organoid models of PDA demonstrate ERBB pathway activation in response to dual MEK/AKT inhibition, and that this feature is absent in non-neoplastic organoids. Furthermore, organoid models predict the limited activity of EGFR specific inhibitors in PDA patients that are also receiving MEK or AKT inhibitors. These models have also been an invaluable resource for probing new dependencies in PDA, and will be discussed. Citation Format: David A. Tuveson. K-RAS dependency in pancreatic cancer. [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 PL03-03.

Abstract 13921: Dysfunctional Bone Morphogenetic Protein Signaling Interlinks With Interleukin-1ß Mediated Inflammation in Pulmonary Arterial Hypertension

Rationale: Bone morphogenetic protein receptor type 2 (BMPR2) mutations are found in heritable and idiopathic pulmonary arterial hypertension but disease penetrance is incomplete implying the necessity for a ‘second hit’. IL-1ß and IL-6 are increased in PAH patients and animal models and are proposed to play a critical role in disease. Objective: To determine pulmonary specific interplay between BMPR2 and IL-1ß signaling by exploring differences between pulmonary artery and aortic smooth muscle cells (PA / Ao SMC) in response to IL-1ß, and determine the effects of reduced BMPR2 expression using in vitro and in vivo disease models. Methods and Results: Microarray analysis of PASMC and AoSMC mRNA show reduced inflammatory pathway activation in response to IL-1ß in PASMC compared with AoSMCs. Pathway analysis identified an exaggerated inflammatory response to IL-1ß in PASMC lacking BMPR2 (siRNA). Significant up-regulation of IL-6, IL-1α and adhesion molecules (>2-fold) shown by array analysis was validated by qPCR. In the absence of BMPR2 a 1.5-fold increase in proliferation was observed in response to IL-1ß that is not present in PASMC with functional BMPR2. To study this further, R899X+/- BMPR2 transgenic mice were fed western diet for six weeks and given daily injections of IL-1ß. Mice were assessed for inflammatory activation and PAH phenotype (catheter/echo). mRNA and protein changes were measured by TaqMan PCR, western blotting and serum ELISA. Mice treated with IL-1ß had significantly higher white blood cell counts (1.7-fold), and showed trends for increased mRNA of VCAM1, IL-1α (whole lung) and significantly raised serum protein levels of IL-6 and OPG matching in vitro data. IL-1ß treated mice had an increase in early pulmonary vascular remodelling and the full assessment of the inflammatory cell infiltrate in R899X+/- mice is currently underway. Conclusion: IL-1ß induces a pulmonary artery specific transcriptome that is altered by suppression of BMPR2 signaling in vitro. In vivo and in vitro IL-1ß drives an exaggerated inflammatory response under conditions of reduced BMPR2 signaling. This research highlights a mechanism by which increased IL-1ß may provide a “second hit” to reduced BMPR2 signaling and stimulate or potentiate the development of PAH.

A biopsychosocial model of fatigue and depression following stroke

Poststroke fatigue (PSF) and poststroke depression (PSD) are both common and difficult sequelae following acute ischemic stroke (AIS). Two main perspectives to explain these sequelae are the biomedical perspective and the psychosocial perspective. Research has shown that PSF and PSD are undoubtedly associated with each other, although each can occur in the absence of the other. However, the nature of the relationship is unclear. For example, do stroke patients become fatigued because of being depressed, or do they become depressed because they are fatigued? Alternatively, is there a bidirectional relationship between these two sequelae, with each influencing the other? We propose a biopsychosocial model of PSF and PSD that supports the AIS-induced immune response and kynurenine pathway activation being related to fatigue but not (directly) to depression. We hypothesize that the risk of developing depression may be reduced if the experience of fatigue is acknowledged, and then addressed accordingly.

Abstract IA41: Role of JAK-STAT pathway activation in MPN pathogenesis and therapeutic response.

Abstract The discovery of JAK2/MPL mutations in the majority of patients with Myeloproliferative Neoplasms (MPN) led to the development of JAK kinase inhibitors. JAK kinase inhibitors, including ruxolitinib, improve patient constitutional symptoms and reduce splenomegaly, but do not significantly reduce mutant allele burden. Chronic exposure to JAK kinase inhibitors results in JAK inhibitor persistence in cell lines, murine models, and patient samples, which we have shown is due to JAK2-transactivation and persistent JAK-STAT signaling. We have used murine genetic studies show that MPN cells remain dependent on JAK2 expression for proliferation and survival. These data suggest that pharmacologic approaches that better inhibit JAK2 activity in MPN cells may demonstrate increased efficacy in vivo. All JAK inhibitors in clinical development are type I inhibitors that interact with and inhibit the active confirmation of the JAK2 kinase. We hypothesized that novel, type II JAK inhibitors that interact with and inhibit JAK2 in the inactive conformation might retain activity in JAK inhibitor persistent cells and show increased efficacy in murine MPN models. Signaling studies demonstrated that CHZ868 more potently attenuated JAK-STAT signaling in JAK2/MPL-mutant cells, with suppression of JAK2 phosphorylation consistent with a type II mechanism of kinase inhibition. CHZ868 completely suppressed JAK-STAT signaling in type I JAK inhibitor-persistent cells, and prevented heterodimeric activation of JAK2 by JAK1 and TYK2. Most importantly, JAK2/MPL-mutant cells which were insensitive to type I JAK inhibitors remained highly sensitive to CHZ868, demonstrating that type I JAK inhibitor persistence does not confer resistance to type II inhibitors. CHZ868 showed significant activity in murine models of Jak2V617F-induced polycythemia vera, and MPLW515L-mutant MF, with normalization of blood counts, stem/progenitor expansion, spleen weights, and extramedullary hematopoiesis in vivo. Most importantly, CHZ868 resulted in significant reductions of mutant allele burden in these models. In addition, it has not been delineated whether the clinical benefits of JAK inhibitors are solely from target inhibition in MPN cells, or if JAK-STAT inhibition in malignant and non-malignant cells contributes to the therapeutic response. We show that the levels of circulating cytokines are elevated in MF and that cytokine production is reduced with JAK inhibitor treatment. Importantly, aberrant cytokine production in MF emanates from both malignant and non-malignant cells, and that JAK inhibition reduces cytokine production from both tumor and non-tumor populations. Our data suggest inhibition of JAK-STAT signaling in malignant and non-malignant cells is required to improve symptoms, reduce disease severity, and to reduce disease progression. Moreover, optimizing therapies to potently inhibit JAK-STAT signaling in malignant and non-malignant cells are warranted to improve clinical efficacy and outcomes in MPN patients. Citation Format: Sara Meyer, Maria Kleppe, Neha Bhagwat, Priya Koppikar, Minsuk Kwak, Thomas Radimerski, Rong Fan, Ross L. Levine. Role of JAK-STAT pathway activation in MPN pathogenesis and therapeutic response. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA41.

Abstract 1158: Real-time assessment of uptake and utilization of lactate in intact human breast cancer cells using a 1H-NMR-based assay

Abstract Objective: The purpose of this study was to evaluate the feasibility of monitoring lactate uptake and utilization in intact human cancer cells using a dynamic 1H-NMR assay and [3-13C]-lactate. This would provide a simple, real-time method for monitoring enzyme kinetics in cells as an alternative to spectrophotometry - which cannot be applied to intact cells - or dynamic nuclear polarisation-NMR, which requires complex mathematical modelling. The ability to better understand and measure lactate metabolism in cancer cells could be applied to monitor MCT1 inhibition. Methods: 3×10^7 MDA-MB-231 human breast cancer cells were suspended in 400μl serum-free DMEM plus 100μl D2O in a 5mm NMR tube. A mixture of 10mM [3-13C]-lactate combined with varying concentrations of unlabelled 12C-pyruvate (0mM, 5mM or 10mM) was added and proton spectra were acquired every minute for 30 minutes on a 500MHz Bruker spectrometer. For comparison, cells were also incubated with 10mM [3-13C]-pyruvate and either 0mM or 10mM 12C-lactate. Results: The presence of 13C-pyruvate was detected in real-time following addition of [3-13C]-lactate indicating that 13C-label had been exchanged from lactate to pyruvate via the enzyme lactate dehydrogenase. The optimisation of reagent concentrations, however, revealed that this observation was dependent on the addition of unlabelled 12C-pyruvate and the apparent rate of 13C-label exchange was proportional to the concentration of 12C-pyruvate added. The data show that no 13C-pyruvate signal is detected when 10mM 13C-lactate is added alone. However, 6% of the 10mM 13C-lactate is exchanged with 13C-pyruvate when 5mM 12C-pyruvate is added (rate = 1.1×10-3mM s-1). This increases to 12% when the 12C-pyruvate concentration was increased to 10mM (rate = 2.2×10-3 mM s-1). The total pyruvate concentration (12C + 13C-pyruvate) remained constant, which suggests that there is no net production of pyruvate. In comparison, the 13C-pyruvate experiment was less dependent on the addition of unlabelled 12C-lactate for 13C-label exchange to be observed. Conclusion: The fact that unlabelled pyruvate was required for a reaction to be observed following addition of 13C-lactate suggests that the position of the isotopic equilibrium favours the label to remain in the lactate pool. Therefore a large amount of unlabelled pyruvate is required for the exchange process to be observed. This would also explain why the addition of unlabelled lactate was not required in the 13C-pyruvate experiment, since to reach isotopic equilibrium, a large proportion of the 13C-pyruvate needs to immediately exchange with the naturally larger lactate pool. Nevertheless, these findings demonstrate the feasibility of using this 1H-NMR assay to measure [3-13C]-lactate uptake in real-time. This could inform on metabolic pathway activity in response to MCT1 inhibition in intact cells. Citation Format: Emily G. Wholey, Harold G. Parkes, Paul Workman, Martin O. Leach, Mounia Beloueche-Babari. Real-time assessment of uptake and utilization of lactate in intact human breast cancer cells using a 1H-NMR-based assay. [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 1158. doi:10.1158/1538-7445.AM2015-1158