The Ketogenic Diet Induces Epigenetic Changes that Play Key Roles in - PowerPoint PPT Presentation

The Ketogenic Diet Induces Epigenetic Changes that Play Key Roles in Tumour Development Jessica Preston, MRes Clinical Research: Human Nutrition Supervisor: Dr. Nelofer Syed John Fulcher Molecular Neuro-Oncology Laboratory 1

Glioblastoma Multiforme • Glioblastoma multiforme (GBM) is a highly aggressive form of brain cancer Survival Rate of GBM based on MRI of GBM in Right Temporal Lobe Status of Tumour Crossing the Midline Dahlrot RH et al. A population-based study of high-grade Schreiber S. et al . Bilateral posterior RION after concomitant radiochemotherapy gliomas and mutated isocitrate dehydrogenase 1. Int J Clin with temozolomide in a patient with glioblastoma multiforme: a case report. BMC Exp Pathol (2013). Cancer (2010). • Current Treatment includes: Complete surgical resection à chemotherapy with temozolomide + radiotherapy

Failure of Current Therapeutics: Tumour Heterogeneity Temozolomide resistance The Warburg Effect Metabolic Therapy is Universal Necrotic core Metastatic phenotype Therapeutic techniques aimed to target the unique metabolism of Hypoxic Glioblastoma tumour tissue. region Tumour Model Glucose Availability = Tumour Tumour Growth Normoxic relapse region Resistance to radio-therapy Persano L. et al . The Three-Layer Concentric Model of Glioblastoma: Cancer Stem Cells, Microenvironmental Regulation, and Therapeutic Heiden M.G.V et al. Understanding the Warburg Effect: The Metabolic Requirements of Implications. ScientificWorldJournal (2011) Cell Proliferation. Science (2009)

The Ketogenic Diet as a Form of Metabolic Therapy • Macronutrient breakdown of a ketogenic diet • 75% Fat • 25% Protein • Few carbohydrates

The Ketogenic Diet as a Form of Metabolic Therapy • Macronutrient breakdown of a ketogenic diet Blood Glucose and β - • 75% Fat Hydroxybutyrate Concentrations • 25% Protein • Few carbohydrates A.L. Hartman et al . Efficacy of the ketogenic diet in the 6-Hz seizure test, Maschio et al. Brain Tumor-Related Epilepsy. Epilepsia (2008). Melo TM. et al. Neuronal-glial interactions in rats fed a ketogenic diet. Neurochemistry International (2006).

The Ketogenic Diet as a form of Metabolic Therapy • Macronutrient breakdown of a ketogenic diet Blood Glucose and β - Hydroxybutyrate Concentrations • 75% Fat • 25% Protein • Few carbohydrates Ketone Bodies ATP A.L. Hartman et al. Efficacy of the ketogenic diet in the 6-Hz seizure test, Maschio et al. Brain Tumor- Related Epilepsy. Epilepsia. 2008. Melo TM. et al. Neuronal-glial interactions in rats fed a ketogenic diet. Neurochemistry International (2006).

The Ketogenic Diet as a Form of Metabolic Therapy • Macronutrient breakdown of a ketogenic diet Blood Glucose and β - • 75% Fat Hydroxybutyrate Concentrations • 25% Protein • Few carbohydrates A.L. Hartman et al . Efficacy of the ketogenic diet in the 6-Hz seizure test, Maschio et al. Brain Tumor-Related Epilepsy. Epilepsia (2008). Melo TM. et al. Neuronal-glial interactions in rats fed a ketogenic diet. Neurochemistry International (2006).

The Ketogenic Diet as a form of Metabolic Therapy • Macronutrient breakdown of a ketogenic diet Blood Glucose and β -Hydroxybutyrate Concentrations • 75% Fat Blood • 25% Protein Ketones • Few carbohydrates Blood Glucose A.L. Hartman et al. Efficacy of the ketogenic diet in the 6-Hz seizure test, Maschio et al. Brain Tumor- Related Epilepsy. Epilepsia. 2008. Melo TM. Et al. Neuronal-glial interactions in rats fed a ketogenic diet. Neurochemistry International. 2006.

The Ketogenic Diet as a Form of Metabolic Therapy • Macronutrient breakdown of a ketogenic diet Blood Glucose and β - • 75% Fat Hydroxybutyrate Concentrations • 25% Protein • Few carbohydrates A.L. Hartman et al . Efficacy of the ketogenic diet in the 6-Hz seizure test, Maschio et al. Brain Tumor-Related Epilepsy. Epilepsia (2008). Melo TM. et al. Neuronal-glial interactions in rats fed a ketogenic diet. Neurochemistry International (2006).

Ketogenic Diet Increases Life Expectancy of Glioma in vivo Survival Curve GL261 Mouse Glioma Collaborators: Dr. Adrienne Scheck Associate Professor M.G. Abdelwaha et al . The Ketogenic Diet Is an Effective Barrow Neurological Adjuvant to Radiation Therapy for the Treatment of Malignant Institute Glioma. Plos One (2012)

The Ketogenic Diet Potentiates the Effects of Radiation Survival Curve GL261 Mouse Glioma Tumour Bioluminescence Radiation Therapy and Ketogenic Diet Radiation Radiation + KetoCal Only Day 3 Day 18 Day 36 M.G. Abdelwaha et al. The Ketogenic Diet Is an Effective Adjuvant to Radiation Therapy for the Treatment of Malignant Glioma. Plos One (2012).

β -Hydroxybutyrate (BHB) Treatment Reduces Cell Proliferation in vitro Established Cell Lines Primary Cell Lines Ella Qingyu Zeng Julianna Stylianou

BHB Enhances the Effectiveness of Radiation and Temozolomide in vitro Cell Proliferation Cell Proliferation Temozolomide Day 6 and BHB Radiation Day 6 and BHB 2.0 **** 1.5 Untreat **** Un 7 Days 1.5 Da **** 18 Day 1.0 450 nm Da 450 nm * 1.0 * * 0.5 0.5 0.0 0.0 0uM 25uM 50uM Gy0 Gy2 Gy4 Irradiation Dose TMZ Concentration (µM) Untreated Day 6 10mM BHB Sophie Glover Day 18 10mM BHB Julianna Stylianou

What is the Mechanism of Action of the Ketogenic Diet? • Epigenetics changes • microRNA expression • chromatin modifying enzymes Hagood JS. Beyond the Genome: Epigenetic Mechanisms in Lung Remodelling. Physiology (2014).

Global microRNA Microarray: Animal Model Differentially Expressed miRs in Standard Differential miR Expression SD vs. KD vs. Ketogenic Diet Large Fold Change (FC>8) mmu-miR-30a-5p mmu-miR-139-5p mmu-miR-149-5p mmu-let-7e-5p mmu-miR-541-5p mmu-miR-138-5p (138-2) mmu-let-7f-5p mmu-miR-346-5p mmu-miR-128-3p mmu-miR-204-5p mmu-miR-92b-3p mmu-miR-138-5p (138-1) 0. 4.25 8.5 12.75 17. 21.25 Fold Change [Treated v. Control]

microRNA Changes in KD Target Anti-Cancer Pathways WikiPathways 2016 Analysis of Differentially Regulated microRNAs miR Expression in KD Treated vs. Standard Tumour Tissue Unknown changes in 23% Changes in neoplastic tissue KD mirror Specific Gene Targets of Differentially Regulated miRs Changes in healthy tissue KD mirror miR-300 miR-503 55% Let-7a-1 neoplastic tissue miR-99a miR-92b miR-128-1 22% VEGF p53 cMYC BMI1 mTOR WNT Signalling

KD Upregulates miR-138 in vivo and in vitro Sophie Glover Julianna Stylianou

KD Induced Upregulation of miR-138 Targets H2AX miR-138-1 H2AX Untreated 10mM BHB Sophie Glover Hoeller D. et al. Targeting the ubiquitin system in cancer therapy. Julianna Stylianou Nature (2009).

KD Influence Expression of Chromatin Modifying Enzyme Expression in vivo Enzyme Expression Ketogenic vs. Down Regulated Genes in KD vs. SD Standard Diet Dnmt3b Smyd1 Prmt8 Hdac11 Up Regulated Genes in KD vs. SD Qiagen Array

KD Induces Up Regulation of PRMT8 PRMT8 Expression in Healthy Simandi Z . et al . PRMT1 and PRMT8 Regulate Retinoic Acid Dependent Neuronal Differentiation versus Cancerous Tissue with Implications to Neuropathology Stem Cells (2014). 5.0x10 -2 2.5x10 -2 0 Normal Glioma Untreated 10mM BHB

KD Down-regulates PRMT8 Target Genes: CXCR4 and DHFR PRMT8 DHFR Expression in Mouse Model CXCR4 in vitro 1.5 1.5 DHFR CXCR4 Normalized Expression Normalized Expression 1.0 1.0 **** 0.5 0.5 * 0.0 0.0 e e c c Untreated D4 D8 i i M M D D S K 10mM BHB DHFR in vitro CXCR4 DHFR 1.5 Normalized Expression • Chemokine receptor for CXCL12 • Dihydrofolic acid donor 1.0 • Maintenance of GBM • Thymidylate synthesis perivascular niche • Enzyme used for DNA 0.5 • Tied to increased migration synthesis and repair ** 0.0 Untreated D6 D8 10mM BHB Days Treated

Retinoic Acid (RA) and BHB Reduce Cell Proliferation in vitro Retinoid Signalling CXCR4 Protein Arginine Methyl- transferase 8 DHFR A Simadi. Transcriptional and epigenetic reprograming of embryonic stem cell differentiation into neurons by retinoic acid. PhD thesis (2014).

In Conclusion • BHB treatment decreases cell proliferation alone and in combination with: • Radiation therapy • Temozolomide • Retinoic Acid • Changes in epigenetic expression with KD implementation including: microRNAs and chromatin modifying enzymes • microRNA-138 upregulation in the KD may lead to down regulation of H2AX • PRMT8 upregulation in KD may lead to down regulation of • DHFR • CXCR4

Acknowledgments Dr. Nelofer Syed Collaborators Dr. Fernando Abaitua Dr. Adrianne Scheck Barrow Neurological Institute Dr. Combiz Khozoie Phoenix Arizona Dr. Julianna Stylianou Mr. Alex Renziehausen Clinical Team Mr. Richard Perryman Mr. Kevin O’Neal Ms. Julia Pazmandi Consultant Neurosurgeon Ms. Ella Qingyu Zeng Mr. Matt Williams Ms. Sophie Glover Consultant Oncologist Mr. Babar Vaqus Mr. John DeFelice