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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

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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

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SLIDE 2

Glioblastoma Multiforme

  • Glioblastoma multiforme (GBM) is a highly aggressive form of brain cancer
  • Current Treatment includes:

Complete surgical resection à chemotherapy with temozolomide + radiotherapy

Survival Rate of GBM based on Status of Tumour Crossing the Midline

Dahlrot RH et al. A population-based study of high-grade gliomas and mutated isocitrate dehydrogenase 1. Int J Clin Exp Pathol (2013).

MRI of GBM in Right Temporal Lobe

Schreiber S. et al. Bilateral posterior RION after concomitant radiochemotherapy with temozolomide in a patient with glioblastoma multiforme: a case report. BMC Cancer (2010).

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SLIDE 3

Failure of Current Therapeutics: Tumour Heterogeneity

Temozolomide resistance Metastatic phenotype Tumour relapse Resistance to radio-therapy Hypoxic region Normoxic region Necrotic core

Persano L. et al. The Three-Layer Concentric Model of Glioblastoma: Cancer Stem Cells, Microenvironmental Regulation, and Therapeutic

  • Implications. ScientificWorldJournal (2011)

Glioblastoma Tumour Model

The Warburg Effect is Universal Metabolic Therapy

Therapeutic techniques aimed to target the unique metabolism of tumour tissue.

Glucose Availability

= Tumour Growth

Heiden M.G.V et al. Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation. Science (2009)

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SLIDE 4

The Ketogenic Diet as a Form of Metabolic Therapy

  • Macronutrient breakdown of a ketogenic diet
  • 75% Fat
  • 25% Protein
  • Few carbohydrates
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SLIDE 5

The Ketogenic Diet as a Form of Metabolic Therapy

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).

Blood Glucose and β- Hydroxybutyrate Concentrations

  • Macronutrient breakdown of a ketogenic diet
  • 75% Fat
  • 25% Protein
  • Few carbohydrates
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SLIDE 6

The Ketogenic Diet as a form of Metabolic Therapy

  • Macronutrient breakdown of a ketogenic diet
  • 75% Fat
  • 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).

Blood Glucose and β- Hydroxybutyrate Concentrations

Ketone Bodies ATP

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SLIDE 7

The Ketogenic Diet as a Form of Metabolic Therapy

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).

Blood Glucose and β- Hydroxybutyrate Concentrations

  • Macronutrient breakdown of a ketogenic diet
  • 75% Fat
  • 25% Protein
  • Few carbohydrates
slide-8
SLIDE 8

The Ketogenic Diet as a form of Metabolic Therapy

  • Macronutrient breakdown of a ketogenic diet
  • 75% Fat
  • 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.

Blood Ketones Blood Glucose Blood Glucose and β-Hydroxybutyrate Concentrations

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SLIDE 9

The Ketogenic Diet as a Form of Metabolic Therapy

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).

Blood Glucose and β- Hydroxybutyrate Concentrations

  • Macronutrient breakdown of a ketogenic diet
  • 75% Fat
  • 25% Protein
  • Few carbohydrates
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SLIDE 10

Ketogenic Diet Increases Life Expectancy of Glioma in vivo

Survival Curve GL261 Mouse Glioma

M.G. Abdelwaha et al. The Ketogenic Diet Is an Effective Adjuvant to Radiation Therapy for the Treatment of Malignant

  • Glioma. Plos One (2012)

Collaborators:

  • Dr. Adrienne Scheck

Associate Professor Barrow Neurological Institute

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SLIDE 11

The Ketogenic Diet Potentiates the Effects of Radiation

Survival Curve GL261 Mouse Glioma Radiation Therapy and Ketogenic Diet Tumour Bioluminescence

M.G. Abdelwaha et al. The Ketogenic Diet Is an Effective Adjuvant to Radiation Therapy for the Treatment of Malignant Glioma. Plos One (2012).

Day 3 Day 18 Radiation Only Radiation + KetoCal Day 36

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SLIDE 12

β-Hydroxybutyrate (BHB) Treatment Reduces Cell Proliferation in vitro

Established Cell Lines Primary Cell Lines

Ella Qingyu Zeng Julianna Stylianou

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SLIDE 13

Cell Proliferation Radiation Day 6 and BHB

Irradiation Dose 450 nm

Gy0 Gy2 Gy4 0.0 0.5 1.0 1.5

Untreat 7 Days 18 Day

BHB Enhances the Effectiveness of Radiation and Temozolomide in vitro

**** * * Cell Proliferation Temozolomide Day 6 and BHB

TMZ Concentration (µM) 450 nm

0uM 25uM 50uM 0.0 0.5 1.0 1.5 2.0

Un Da Da

Untreated Day 6 10mM BHB Day 18 10mM BHB

**** **** *

Sophie Glover Julianna Stylianou

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SLIDE 14

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).

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SLIDE 15

Global microRNA Microarray: Animal Model

Fold Change [Treated v. Control]

0. 4.25 8.5 12.75 17. 21.25

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)

Differential miR Expression SD vs. KD Large Fold Change (FC>8) Differentially Expressed miRs in Standard

  • vs. Ketogenic Diet
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SLIDE 16

Changes in KD mirror healthy tissue Changes in KD mirror neoplastic tissue Unknown changes in neoplastic tissue

miR Expression in KD Treated

  • vs. Standard Tumour Tissue

microRNA Changes in KD Target Anti-Cancer Pathways

WikiPathways 2016 Analysis of Differentially Regulated microRNAs Specific Gene Targets of Differentially Regulated miRs miR-300 p53 miR-99a mTOR Let-7a-1 cMYC miR-92b WNT Signalling miR-503 VEGF miR-128-1 BMI1 55% 22% 23%

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SLIDE 17

KD Upregulates miR-138 in vivo and in vitro

Sophie Glover Julianna Stylianou

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SLIDE 18

KD Induced Upregulation of miR-138 Targets H2AX

miR-138-1 H2AX

Hoeller D. et al. Targeting the ubiquitin system in cancer therapy. Nature (2009).

Untreated 10mM BHB

Sophie Glover Julianna Stylianou

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SLIDE 19

KD Influence Expression of Chromatin Modifying Enzyme Expression in vivo

Enzyme Expression Ketogenic vs. Standard Diet

Prmt8 Hdac11 Smyd1 Dnmt3b

Down Regulated Genes in KD vs. SD Up Regulated Genes in KD vs. SD

Qiagen Array

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SLIDE 20

KD Induces Up Regulation of PRMT8

Simandi Z. et al. PRMT1 and PRMT8 Regulate Retinoic Acid Dependent Neuronal Differentiation with Implications to Neuropathology Stem Cells (2014).

Untreated 10mM BHB

Normal Glioma PRMT8 Expression in Healthy versus Cancerous Tissue

5.0x10-2 2.5x10-2

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SLIDE 21

DHFR Expression in Mouse Model

Normalized Expression

S D M i c e K D M i c e 0.0 0.5 1.0 1.5

**** CXCR4 in vitro

Normalized Expression

Untreated D4 D8 0.0 0.5 1.0 1.5

*

KD Down-regulates PRMT8 Target Genes: CXCR4 and DHFR

10mM BHB

DHFR in vitro

Days Treated Normalized Expression

Untreated D6 D8 0.0 0.5 1.0 1.5

**

10mM BHB

DHFR

  • Dihydrofolic acid donor
  • Thymidylate synthesis
  • Enzyme used for DNA

synthesis and repair

PRMT8

DHFR CXCR4

CXCR4

  • Chemokine receptor for CXCL12
  • Maintenance of GBM

perivascular niche

  • Tied to increased migration
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SLIDE 22

Retinoic Acid (RA) and BHB Reduce Cell Proliferation in vitro

CXCR4 DHFR

Protein Arginine Methyl- transferase 8

A Simadi. Transcriptional and epigenetic reprograming of embryonic stem cell differentiation into neurons by retinoic acid. PhD thesis (2014).

Retinoid Signalling

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SLIDE 23

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
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SLIDE 24

Acknowledgments

  • Dr. Nelofer Syed
  • Dr. Fernando Abaitua
  • Dr. Combiz Khozoie
  • Dr. Julianna Stylianou
  • Mr. Alex Renziehausen
  • Mr. Richard Perryman
  • Ms. Julia Pazmandi
  • Ms. Ella Qingyu Zeng
  • Ms. Sophie Glover
  • Mr. John DeFelice

Collaborators

  • Dr. Adrianne Scheck

Barrow Neurological Institute Phoenix Arizona

Clinical Team

  • Mr. Kevin O’Neal

Consultant Neurosurgeon

  • Mr. Matt Williams

Consultant Oncologist

  • Mr. Babar Vaqus