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Supplementary Table 6 from Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

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Supplementary Figure 5 from Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

Supplementary Table 4 from Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

Supplementary Table 2 from Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

Supplementary Figure 2 from Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

Supplementary Figure 1 from Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

Supplementary Figure 4 from Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

Supplementary References from Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

Supplementary Figure 6 from Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

Supplementary Table 7 from Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

Supplementary Table 1 from Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

Supplementary Table 3 from Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

Supplementary Table 5 from Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

Supplementary Figure 7 from Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

Supplementary Figure 3 from Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

Supplementary Table and Figure Legends from Transcriptional Pathway Signatures Predict MEK Addiction and Response to Selumetinib (AZD6244)

DNA methylation of gene promoter regions represses transcription and is a mechanism via which environmental risk factors could affect cells during development in individuals at risk for schizophrenia. We investigated DNA methylation in patient-derived cells that might shed light on early development in schizophrenia. Induced pluripotent stem cells may reflect a “ground state” upon which developmental and environmental influences would be minimal. Olfactory neurosphere-derived cells are an adult-derived neuro-ectodermal stem cell modified by developmental and environmental influences. Fibroblasts provide a non-neural control for life-long developmental and environmental influences. Genome-wide profiling of DNA methylation and gene expression was done in these three cell types from the same individuals. All cell types had distinct, statistically significant schizophrenia-associated differences in DNA methylation and linked gene expression, with Gene Ontology analysis showing that the differentially affected genes clustered in networks associated with cell growth, proliferation, and movement, functions known to be affected in schizophrenia patient-derived cells. Only five gene loci were differentially methylated in all three cell types. Understanding the role of epigenetics in cell function in the brain in schizophrenia is likely to be complicated by similar cell type differences in intrinsic and environmentally induced epigenetic regulation., Epigenetics: A genome-wide picture in patient-derived cells Schizophrenia-associated differences in the DNA methylation status of patient-derived cells suggest it could affect early brain development. Mechanisms that control gene expression without altering the genetic code, such as DNA methylation, could explain how environmental risk factors contribute to schizophrenia in genetically susceptible individuals. Alan Mackay-Sim and colleagues from Griffith University, Australia, carried out genome-wide comparisons of DNA methylation in induced pluripotent stem (iPS) cells, olfactory neurosphere-derived cells and fibroblasts from patients and controls. Differences in the DNA methylation pattern between patient and control iPS cells, which could reflect what happens in the embryo, suggest a disease-associated effect very early on in development. Only five genes were differentially methylated in all three patient-derived cell types compared to controls. None of these genes has previously been associated with schizophrenia and may represent new targets for future research.

Selumetinib (AZD6244, ARRY-142886) is a selective, non–ATP-competitive inhibitor of mitogen-activated protein/extracellular signal–regulated kinase kinase (MEK)-1/2. The range of antitumor activity seen preclinically and in patients highlights the importance of identifying determinants of response to this drug. In large tumor cell panels of diverse lineage, we show that MEK inhibitor response does not have an absolute correlation with mutational or phospho-protein markers of BRAF/MEK, RAS, or phosphoinositide 3-kinase (PI3K) activity. We aimed to enhance predictivity by measuring pathway output through coregulated gene networks displaying differential mRNA expression exclusive to resistant cell subsets and correlated to mutational or dynamic pathway activity. We discovered an 18-gene signature enabling measurement of MEK functional output independent of tumor genotype. Where the MEK pathway is activated but the cells remain resistant to selumetinib, we identified a 13-gene signature that implicates the existence of compensatory signaling from RAS effectors other than PI3K. The ability of these signatures to stratify samples according to functional activation of MEK and/or selumetinib sensitivity was shown in multiple independent melanoma, colon, breast, and lung tumor cell lines and in xenograft models. Furthermore, we were able to measure these signatures in fixed archival melanoma tumor samples using a single RT-qPCR–based test and found intergene correlations and associations with genetic markers of pathway activity to be preserved. These signatures offer useful tools for the study of MEK biology and clinical application of MEK inhibitors, and the novel approaches taken may benefit other targeted therapies. Cancer Res; 70(6); 2264–73

Melanocytes respond to UVR not only by producing melanin, but also by proliferating. This is essentially a protective response. We have studied the melanocyte proliferative response after a single UVR exposure to neonatal mice. At 3 days post-UVR in wild-type neonates we observed a marked melanocyte activation not seen in adults. Melanocytes migrated to the epidermal basal layer, their numbers peaking at 3-5 days after UVR then diminishing. They appeared to emanate from the hair follicle, migrating to the epidermis via the outer root sheath. In melanoma-prone mice with melanocyte-specific overexpression of Hras(G12V), basal layer melanocytes were increased in size and dendricity compared to UVR-treated wild-type mice. Melanocytes in mice carrying a pRb pathway cell-cycle defect (oncogenic Cdk4(R24C)) did not show an enhanced response to UVR such as those carrying Hras(G12V). The exquisite sensitivity to UVR-induced proliferation and migration that characterizes neonatal mouse melanocytes may partly explain the utility of this form of exposure for inducing melanoma in mice that carry oncogenic mutations.

Parkinson's disease is a complex age-related neurodegenerative disorder. Approximately 90% of Parkinson's disease cases are idiopathic, of unknown origin. The aetiology of Parkinson's disease is not fully understood but increasing evidence implies a failure in fundamental cellular processes including mitochondrial dysfunction and increased oxidative stress. To dissect the cellular events underlying idiopathic Parkinson's disease, we use primary cell lines established from the olfactory mucosa of Parkinson's disease patients. Previous metabolic and transcriptomic analyses identified deficiencies in stress response pathways in patient-derived cell lines. The aim of this study was to investigate whether these deficiencies manifested as increased susceptibility, as measured by cell viability, to a range of extrinsic stressors. We identified that patient-derived cells are more sensitive to mitochondrial complex I inhibition and hydrogen peroxide induced oxidative stress, than controls. Exposure to low levels (50 nM) of rotenone led to increased apoptosis in patient-derived cells. We identified an endogenous deficit in mitochondrial complex I in patient-derived cells, but this did not directly correlate with rotenone-sensitivity. We further characterized the sensitivity to rotenone and identified that it was partly associated with heat shock protein 27 levels. Finally, transcriptomic analysis following rotenone exposure revealed that patient-derived cells express a diminished response to rotenone-induced stress compared with cells from healthy controls. Our cellular model of idiopathic Parkinson's disease displays a clear susceptibility phenotype to mitochondrial stress. The determination of molecular mechanisms underpinning this susceptibility may lead to the identification of biomarkers for either disease onset or progression.

In the field of disease modeling, induced pluripotent stem cells (iPSCs) have become an appealing choice, especially for diseases that do not have an animal model. They can be generated from patients with known clinical features and compared with cells from healthy controls to identify the biological bases of disease. This study was undertaken to determine the variability in iPSC lines derived from different individuals, with the aim of determining criteria for selecting iPSC lines for disease models. We generated and characterized 18 iPSC lines from eight donors and considered variability at three levels: (a) variability in the criteria that define iPSC lines as pluripotent cells, (b) variability in cell lines from different donors, and (c) variability in cell lines from the same donor. We found that variability in transgene expression and pluripotency marker levels did not prevent iPSCs from fulfilling all other criteria for pluripotency, including teratoma formation. We found low interindividual and interclonal variability in iPSCs that fulfilled the most stringent criteria for pluripotency, with very high correlation in their gene expression profiles. Interestingly, some cell lines exhibited reprogramming instability, spontaneously regressing from a fully to a partially reprogrammed state. This was associated with a low percentage of cells expressing the pluripotency marker stage-specific embryonic antigen-4. Our study shows that it is possible to define a similar “ground state” for each cell line as the basis for making patient versus control comparisons, an essential step in order to identify disease-associated variability above individual and cell line variability.

Background: Without appropriate cellular models the etiology of idiopathic Parkinson's disease remains unknown. We recently reported a novel patient-derived cellular model generated from biopsies of the olfactory mucosa (termed olfactory neurosphere-derived (hONS) cells) which express functional and genetic differences in a disease-specific manner. Transcriptomic analysis of Patient and Control hONS cells identified the NRF2 transcription factor signalling pathway as the most differentially expressed in Parkinson's disease.\ud \ud Results: We tested the robustness of our initial findings by including additional cell lines and confirmed that hONS cells from Patients had 20% reductions in reduced glutathione levels and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carbo​xymethoxyphenyl)-2-(4-sulfophenyl)-2H-te​trazolium,inner salt] metabolism compared to cultures from healthy Control donors. We also confirmed that Patient hONS cells are in a state of oxidative stress due to higher production of H2O2 than Control cultures. siRNA-mediated ablation of NRF2 in Control donor cells decreased both total glutathione content and MTS metabolism to levels detected in cells from Parkinson's Disease patients. Conversely, and more importantly, we showed that activation of the NRF2 pathway in Parkinson's disease hONS cultures restored glutathione levels and MTS metabolism to Control levels. Paradoxically, transcriptomic analysis after NRF2 pathway activation revealed an increased number of differentially expressed mRNAs within the NRF2 pathway in L-SUL treated Patient-derived hONS cells compared to L-SUL treated Controls, even though their metabolism was restored to normal. We also identified differential expression of the PI3K/AKT signalling pathway, but only post-treatment.\ud \ud Conclusions: Our results confirmed NRF2 as a potential therapeutic target for Parkinson's disease and provided the first demonstration that NRF2 function was inducible in Patient-derived cells from donors with uniquely varied genetic backgrounds. However, our results also demonstrated that the response of PD patient-derived cells was not co-ordinated in the same way as in Control cells. This may be an important factor when developing new therapeutics.

SUMMARY There is a pressing need for patient-derived cell models of brain diseases that are relevant and robust enough to produce the large quantities of cells required for molecular and functional analyses. We describe here a new cell model based on patient-derived cells from the human olfactory mucosa, the organ of smell, which regenerates throughout life from neural stem cells. Olfactory mucosa biopsies were obtained from healthy controls and patients with either schizophrenia, a neurodevelopmental psychiatric disorder, or Parkinson’s disease, a neurodegenerative disease. Biopsies were dissociated and grown as neurospheres in defined medium. Neurosphere-derived cell lines were grown in serum-containing medium as adherent monolayers and stored frozen. By comparing 42 patient and control cell lines we demonstrated significant disease-specific alterations in gene expression, protein expression and cell function, including dysregulated neurodevelopmental pathways in schizophrenia and dysregulated mitochondrial function, oxidative stress and xenobiotic metabolism in Parkinson’s disease. The study has identified new candidate genes and cell pathways for future investigation. Fibroblasts from schizophrenia patients did not show these differences. Olfactory neurosphere-derived cells have many advantages over embryonic stem cells and induced pluripotent stem cells as models for brain diseases. They do not require genetic reprogramming and they can be obtained from adults with complex genetic diseases. They will be useful for understanding disease aetiology, for diagnostics and for drug discovery.