<p>Supplementary Table S5. RPPA analysis of phosphorylated and total protein levels in PC9 and NCI-H1975 AZD9291 resistant populations compared to respective parental cells.</p>

Supplementary Figure Legend. Legends for Supplementary Figures S1-S6.

Supplementary Tables S1-S4. Generation of resistant cell populations (S1); Small molecule inhibitors (S2); IC50 (µM) values from cell growth inhibition assays comparing compound sensitivity between parental and resistant cell populations (S3); Genetic analysis of resistant cell populations (S4).

Supplementary Methods and References. Description of additional methods and procedures used in the study. Also includes Supplementary References.

<p>Supplementary Figures S1-S6. Comparison of genetic alterations across multiple populations resistant to AZD9291 and other EGFR TKIs (S1); Treatment of resistant populations with AZD9291 (S2); Detection and Validation of a novel NRAS E63K mutation (S3); Lysates were prepared from parental PC9 and resistant populations analysed for levels of total and phosphorylated ERK, NRAS and KRAS by western blot (S4); The novel NRAS E63K mutation is an activating mutation that when expressed enhances resistance to cell growth inhibition by gefitinib or AZD9291 in EGFRm cell lines (S5); In vitro combination of AZD9291 with selumetinib induces more profound phenotype inhibition (S6).</p>

The insulin/insulin-like growth factor 1 (IGF1) signaling pathways are implicated in longevity and in progression of Alzheimer's disease. Previously, we showed that insulin-like growth factor 1 receptor (IGF1R) and downstream signaling transcripts are reduced in astrocytes in human brain with progression of Alzheimer's neuropathology and developed a model of IGF1 signaling impairment in human astrocytes using an IGF1R-specific monoclonal antibody, MAB391. Here, we have established a novel human astrocyte-neuron co-culture system to determine whether loss of astrocytic IGF1R affects their support for neurons. Astrocyte-neuron co-cultures were developed using human primary astrocytes and differentiated Lund Human Mesencephalic Cells (LUHMES). Neurite outgrowth assays, performed to measure astrocytic support for neurons, showed astrocytes provided contact-mediated support for neurite outgrowth. Loss of IGF1R did not affect neurite outgrowth under control conditions but when challenged with hydrogen peroxide IGF1R-impaired astrocytes were less able to protect LUHMES. To determine how loss of IGF1R affects neuronal support MAB391-treated astrocytes were FACS sorted from GFP-LUHMES and their transcriptomic profile was investigated using microarrays. Changes in transcripts involved in astrocyte energy metabolism were identified, particularly NDUFA2 and NDUFB6, which are related to complex I assembly. Loss of complex I activity in MAB391-treated astrocytes validated these findings. In conclusion, reduced IGF1 signaling in astrocytes impairs their support for neurons under conditions of stress and this is associated with defects in the mitochondrial respiratory chain in astrocytes.

Astrocytes have essential roles in the central nervous system and are also implicated in the pathogenesis of neurodegenerative disease. Forming non-overlapping domains, astrocytes are highly complex cells. Immunohistochemistry to a variety of proteins can be used to study astrocytes in tissue, labelling different cellular components and sub-populations, including glial fibrillary acidic protein, ALDH1L1, CD44, NDRG2 and amino acid transporters, but none of these labels the entire astrocyte population. Increasing heterogeneity is recognized in the astrocyte population, a complexity that is relevant both to their normal function and pathogenic roles. They are involved in neuronal support, as active components of the tripartite synapse and in cell interactions within the neurovascular unit (NVU), where they are essential for blood-brain barrier maintenance and neurovascular coupling. Astrocytes change with age, and their responses may modulate the cellular effects of neurodegenerative pathologies, which alone do not explain all of the variance in statistical models of neurodegenerative dementias. Astrocytes respond to both the neurofibrillary tangles and plaques of Alzheimer's disease, to hyperphosphorylated tau and Aβ, eliciting an effect which may be neuroprotective or deleterious. Not only astrocyte hypertrophy, in the form of gliosis, occurs, but also astrocyte injury and atrophy. Loss of normal astrocyte functions may contribute to reduced support for neurones and dysfunction of the NVU. Understanding how astrocytes contribute to dementia requires an understanding of the underlying heterogeneity of astrocyte populations, and the complexity of their responses to pathology. Enhancing the supportive and neuroprotective components of the astrocyte response has potential translational applications in therapeutic approaches to dementia.

The accumulation of reactive oxygen species leading to oxidative damage and cell death plays an important role in a number of neurodegenerative disorders. FOXO3a, the main isoform of FOXO transcription factors, mediates the cellular response to oxidative stress by regulating the expression of genes involved in DNA repair and glutamine metabolism, including glutamine synthetase (GS). Immunohistochemical investigation of the population-based neuropathology cohort of the Medical Research Council's Cognitive Function and Ageing Study (MRC CFAS) demonstrates that nuclear retention of FOXO3a significantly correlates with a DNA damage response and with GS expression by astrocytes. Furthermore, we show that GS expression correlates with increasing Alzheimer-type pathology in this ageing cohort. Our findings suggest that in response to oxidative stress, the nuclear retention of FOXO3a in astrocytes upregulates expression of GS as a neuroprotective mechanism. However, the activity of GS may be compromised by increasing levels of oxidative stress in the ageing brain resulting in dysfunctional enzyme activity, neuronal excitotoxic damage and cognitive impairment.

AbstractResistance to targeted EGFR inhibitors is likely to develop in EGFR-mutant lung cancers. Early identification of innate or acquired resistance mechanisms to these agents is essential to direct development of future therapies. We describe the detection of heterogeneous mechanisms of resistance within populations of EGFR-mutant cells (PC9 and/or NCI-H1975) with acquired resistance to current and newly developed EGFR tyrosine kinase inhibitors, including AZD9291. We report the detection of NRAS mutations, including a novel E63K mutation, and a gain of copy number of WT NRAS or WT KRAS in cell populations resistant to gefitinib, afatinib, WZ4002, or AZD9291. Compared with parental cells, a number of resistant cell populations were more sensitive to inhibition by the MEK inhibitor selumetinib (AZD6244; ARRY-142886) when treated in combination with the originating EGFR inhibitor. In vitro, a combination of AZD9291 with selumetinib prevented emergence of resistance in PC9 cells and delayed resistance in NCI-H1975 cells. In vivo, concomitant dosing of AZD9291 with selumetinib caused regression of AZD9291-resistant tumors in an EGFRm/T790M transgenic model. Our data support the use of a combination of AZD9291 with a MEK inhibitor to delay or prevent resistance to AZD9291 in EGFRm and/or EGFRm/T790M tumors. Furthermore, these findings suggest that NRAS modifications in tumor samples from patients who have progressed on current or EGFR inhibitors in development may support subsequent treatment with a combination of EGFR and MEK inhibition. Cancer Res; 75(12); 2489–500. ©2015 AACR.

AIMS:Oxidative damage and an associated DNA damage response (DDR) are evident in mild cognitive impairment and early Alzheimer's disease, suggesting that neuronal dysfunction resulting from oxidative DNA damage may account for some of the cognitive impairment not fully explained by Alzheimer-type pathology. METHODS:Frontal cortex (Braak stage 0-II) was obtained from the Medical Research Council's Cognitive Function and Ageing Study cohort. Neurones were isolated from eight cases (four high and four low DDR) by laser capture microdissection and changes in the transcriptome identified by microarray analysis. RESULTS:Two thousand three hundred seventy-eight genes were significantly differentially expressed (1690 up-regulated, 688 down-regulated, P < 0.001) in cases with a high neuronal DDR. Functional grouping identified dysregulation of cholesterol biosynthesis, insulin and Wnt signalling, and up-regulation of glycogen synthase kinase 3β. Candidate genes were validated by quantitative real-time polymerase chain reaction. Cerebrospinal fluid levels of 24(S)-hydroxycholesterol associated with neuronal DDR across all Braak stages (rs  = 0.30, P = 0.03). CONCLUSIONS:A persistent neuronal DDR may result in increased cholesterol biosynthesis, impaired insulin and Wnt signalling, and increased GSK3β, thereby contributing to neuronal dysfunction independent of Alzheimer-type pathology in the ageing brain.

BACKGROUND:The insulin/IGF1 signalling (IIS) pathways are involved in longevity regulation and are dysregulated in neurons in Alzheimer's disease (AD). We previously showed downregulation in IIS gene expression in astrocytes with AD-neuropathology progression, but IIS in astrocytes remains poorly understood. We therefore examined the IIS pathway in human astrocytes and developed models to reduce IIS at the level of the insulin or the IGF1 receptor (IGF1R). RESULTS:We determined IIS was present and functional in human astrocytes by immunoblotting and showed astrocytes express the insulin receptor (IR)-B isoform of Ir. Immunocytochemistry and cell fractionation followed by western blotting revealed the phosphorylation status of insulin receptor substrate (IRS1) affects its subcellular localisation. To validate IRS1 expression patterns observed in culture, expression of key pathway components was assessed on post-mortem AD and control tissue using immunohistochemistry. Insulin signalling was impaired in cultured astrocytes by treatment with insulin + fructose and resulted in decreased IR and Akt phosphorylation (pAkt S473). A monoclonal antibody against IGF1R (MAB391) induced degradation of IGF1R receptor with an associated decrease in downstream pAkt S473. Neither treatment affected cell growth or viability as measured by MTT and Cyquant® assays or GFAP immunoreactivity. DISCUSSION:IIS is functional in astrocytes. IR-B is expressed in astrocytes which differs from the pattern in neurons, and may be important in differential susceptibility of astrocytes and neurons to insulin resistance. The variable presence of IRS1 in the nucleus, dependent on phosphorylation pattern, suggests the function of signalling molecules is not confined to cytoplasmic cascades. Down-regulation of IR and IGF1R, achieved by insulin + fructose and monoclonal antibody treatments, results in decreased downstream signalling, though the lack of effect on viability suggests that astrocytes can compensate for changes in single pathways. Changes in signalling in astrocytes, as well as in neurons, may be important in ageing and neurodegeneration.

Abstract First- and second-generation EGFR TKIs are established first line therapy for patients with NSCLC with activating mutations in EGFR. Unfortunately, patients ultimately develop disease progression with acquisition of a second-site EGFR T790M mutation in more than half of cases. This has led to the development of third generation EGFR TKIs which inhibit both the EGFRm+ and T790M mutations in preclinical models and are showing activity in TKI-resistant patients in Phase I studies. Despite the potential improvements brought by third generation EGFR-TKIs, advanced EGFRm+ tumor cells will still remain highly adaptable and the inevitability of further resistance will limit the effectiveness of these drugs. As such, the identification of resistance mechanisms to these agents is essential to guide future therapeutic strategies and identify novel:novel combinations. To interrogate resistance to AZD9291, we have generated panels of EGFRm+ cell lines resistant to gefitinib (first generation TKI) and EGFRm+ and EGFRm+/T790M cell lines resistant to afatinib and AZD9291 (second and third generation TKIs, respectively). Subsequently, we characterised the cell lines using a variety of molecular profiling techniques including Next Generation Sequencing (NGS), Affymetrix gene expression analysis, and phenotypic profiling following pharmacological modulation by small molecule inhibitors of canonical signaling pathways. The effects on cell survival across the range of resistant models by a panel of pathway inhibitors indicated that resistance to the EGFR inhibitors, in particular AZD9291, is frequently associated with increased sensitivity to selumetinib (AZD6244; ARRY-142886) (MEK1/2 inhibitor), suggesting that ERK signaling is commonly reactivated to circumvent inhibition of the EGFR pathway. Molecular characterisation of the panel of cell lines suggests a range of different resistance mechanisms may be responsible for reactivation of ERK signaling, including a decrease in negative regulators of ERK such as DUSP6 or an epithelial to mesenchymal transition consistent with previous observations. Collectively, these data suggest that combining an EGFR TKI with a MEK inhibitor could prevent or delay resistance and drive superior duration of benefit from these agents. Citation Format: Cath Eberlein, Laura Ratcliffe, Lucy O'Brien, Katie Al-Khadimi, Henry Brown, Paul Fisher, Daniel Stetson, Zhongwu Lai, Gayle Marshall, Claire Barnes, Kenneth Thress, Brian Dougherty, William Pao, Darren Cross. Investigating resistance to AZD9291. [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 1722. doi:10.1158/1538-7445.AM2014-1722

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development by activating 3 receptor tyrosine kinases (RTKs), VEGFR-1, -2, and -3, and by binding to coreceptors such as neuropilin-1 (NRP-1). We investigated how different VEGF-A isoforms, in particular VEGF-A(165)a and VEGF-A(165)b, control the balance between VEGFR-2 recycling, degradation, and signaling. Stimulation of cells with the NRP-1-binding VEGF-A(165)a led to sequential NRP-1-mediated VEGFR-2 recycling through Rab5, Rab4, and Rab11 vesicles. Recycling was accompanied by dephosphorylation of VEGFR-2 between Rab4 and Rab11 vesicles and quantitatively and qualitatively altered signal output. In cells stimulated with VEGF-A(165)b, an isoform unable to bind NRP-1, VEGFR-2 bypassed Rab11 vesicles and was routed to the degradative pathway specified by Rab7 vesicles. Deletion of the GIPC (synectin) binding motif of NRP-1 prevented transition of VEGFR-2 through Rab11 vesicles and attenuated signaling. Coreceptor engagement was specific for VEGFR-2 because EGFR recycled through Rab11 vesicles in the absence of known coreceptors. Our data establish a distinct role of NRP-1 in VEGFR-2 signaling and reveal a general mechanism for the function of coreceptors in modulating RTK signal output.