Of Epigenetic Modulation by Valproic Acid in Traumatic Brain Injury – What We

TITLE PAGE

Title: The ‘Omics’ of Epigenetic Modulation by Valproic Acid in Traumatic Brain Injury – What We

Know and What the Future Holds

Short title: The ‘Omics’ of Valproic Acid treatment for TBI

Authors:

Umar F. Bhatti, MD; Aaron M. Williams, MD; Patrick E. Georgoff, MD; Hasan B. Alam, MD

Affiliations:

Department of Surgery, University of Michigan, Ann Arbor, MI, USA.

Address for correspondence:

Hasan B. Alam, MD

Norman Thompson Professor of Surgery, and Head of General Surgery

University of Michigan Hospital

2920 Taubman Center/5331

University of Michigan Hospital

1500 E. Medical Center Drive

This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/prca.201900068.

Ann Arbor, MI 48109-5331 [email protected]

Abbreviations:

VPA = Valproic acid

HAT = Histone Acetylase

HDAC = Histone Deacetylase

TBI = Traumatic Brain Injury

HS = Hemorrhagic Shock

PBMC = Peripheral Blood Mononuclear Cells

NEFL = Neurofilament Light

ELISA = Enzyme-linked Immunosorbent Assay

PCR = Polymerase Chain Reaction

LINCS = Library of Integrated Network-based Cellular Signatures

Keywords: epigenetic modulation, valproic acid, omics, traumatic brain injury, clinical trial

No. of words: 2485 words

ABSTRACT

Traumatic brain injury is a heterogeneous injury that is a major cause of morbidity and mortality worldwide. Epigenetic modulation via acetylation by valproic acid has shown promise as an effective pharmacological treatment for TBI; however, the mechanisms by which it improves clinical outcomes are not well-described. In recent years, omics technologies have emerged as a promising strategy to detect molecular changes at the cellular level. This review highlights the use of omics technologies in advancing the understanding of epigenetic modulation by VPA in TBI. It also describes the future role of omics techniques in developing a point of care test to guide patient selection for VPA administration.

Introduction

Traumatic brain injury remains a major health problem around the world.[1] It is the leading cause of morbidity and mortality in young adults. [2] In the United States alone, TBI affects nearly 1.5 to 2.0 million each year,[3] resulting in approximately 250,000 hospitalizations. As a result, 50,000

Americans die yearly, and 90,000 experience long-term disability. [4] Furthermore, according to the

Center for Disease Control and Prevention, the estimated economic cost of TBI in 2010 was approximately $76.5 billion. [5] Despite recent advances in medical care, there is a lack of effective pharmacologic treatment options for TBI. However, valproic acid, a histone deacetylase inhibitor, has shown promise in animal models of TBI.[6] [7]

The Nature of Traumatic Brain Injury

TBI is a heterogeneous injury.[8] The many classification systems for TBI include those based on injury severity,[9] anatomy of the injured region,[10] mechanism of injury,[11]pathophysiology.[12] and prognostic modelling. [13] Although previously considered to be a single event, TBI is a disease process that starts with the inciting injury and may progress secondary to insults such as hypoxia and

hypotension. TBI may even progress many years after the primary injury. [14] Due to its heterogeneous nature, development of pharmacologic interventions for TBI is challenging. [15] [16]

Role of Epigenetic Modulation in TBI

Recently, acetylation of proteins (histone and non-histone) has gained attention as a therapeutic strategy for a variety of diseases, including cancer and trauma.[17] Histone acetylase and histone deacetylase (HDAC) are two enzymes that influence the acetylation status of a cell.[18] An imbalance between these key enzymes may result in modification of critical activator and silencer proteins involved in gene transcription. In an inactive form, the chromatin (complex of DNA and proteins within nucleus) is in a condensed conformation. Acetylation of these proteins promotes chromatin relaxation, allowing transcription factors to bind to chromosomes and thereby promoting gene expression.[19]

Valproic Acid (VPA) – A Histone Deacetylase Inhibitor

VPA, an FDA-approved drug for epilepsy, is an inhibitor of the enzyme histone deacetylase

(HDAC).[20] By inhibiting HDAC and promoting acetylation of histones, VPA can alter gene expression. This action of VPA has proven beneficial in a variety of pathological states, including

TBI.[18] The downstream effects of VPA are considered to be ‘pro-survival’ as it can exploit innate pathways that promote cell growth and differentiation and inhibit apoptosis and inflammation. [21]

Several pre-clinical studies have demonstrated VPA’s effects in TBI with and without haemorrhage [22] [23] and spinal cord injuries[24]. Swine models of TBI have been used to demonstrate that a single large dose of VPA (150 mg/kg) can decrease brain lesion size and swelling, minimize neurologic injury, and shorten the time to recovery.[6] Because of its robust effects in animals, VPA is

now being tested in humans. A phase I dose-escalation trial was recently finished, demonstrating the safety of high doses of VPA in healthy humans (ClinicalTrials.gov identifier NCT01951560). [25]

The Emergence of ‘Omics’ Technologies

Because of its marked effects in the treatment of TBI in preclinical studies, understanding

VPA’s mechanisms of action in trauma has become an area of interest. Traditional laboratory techniques like western blot, enzyme-linked immune-sorbent assay (ELISA) and polymerase chain reaction (PCR) have proven to be limited in many ways. For example, western blot can only be performed if the protein of interest has a specific primary antibody available. Even if an antibody to the target protein is available, it may have off-target effects which may decrease the accuracy of results. [26] Antibody cross-reactivity may not only affect the results of western blot, but also of

ELISA. [27] Also, when proteins are absorbed to plastic surfaces, the epitopes of these proteins can get denatured resulting in alterations in antigens, which is known as the surface effect. Such changes can limit the utility of enzyme immunoassays.[28] On the other hand, omics technologies have emerged as a promising strategy to discover biological changes. [29] Knowledge of the molecular and cellular changes has afforded correlation of cellular events and effects.

Omics technologies, including next-generation DNA and RNA sequencing, proteomics, and metabolomics, have enhanced our understanding of disease states, including TBI. In rats subjected to lateral fluid-percussion injury, peri-lesional cortex demonstrated an increase in transcription factors

Pax6, Tp73, Cebpd, and Myb. Library of Integrated Network-based Cellular Signatures analysis has shown that certain drugs already in clinical use can modulate the expression of these key regulatory transcription factors.[30] Another study that used fresh human brain biopsies for mass-spectrometry

(MS)-based proteomic analysis showed that there are several proteomic alterations in these brains following TBI. This study also demonstrated that these alterations were more pronounced in patients

with widespread axonal injury when compared with focal injury, which may result in a rapid induction of secondary brain injury in these severely injured brains.[31] While the pathological changes associated with TBI have been studied extensively, a gap in knowledge remains as to how therapeutic interventions, such as VPA, may improve clinical outcomes following TBI. We, therefore, decided to utilize these omics techniques to advance the understanding of VPA’s mechanisms of action for the treatment of TBI. Here, we summarize our findings.

Overview of our TBI model

All the TBI models used in our experiments are highly reproducible and clinically relevant.[6] [7]

In summary, female Yorkshire swine are acquired from Michigan State university, East Lansing,

Michigan, USA, and allowed an acclimation period of five days. On the day of experiment, swine are anesthetized, and a transdermal fentanyl patch is placed for pain control. Prophylactic antibiotics are administered via peripheral venous catheters. Femoral vessels are cannulated for haemorrhage and blood pressure monitoring. A craniotomy is performed to expose the dura. A stereotactic computer- controlled cortical impact (CCI) device that is developed by the University of Michigan Innovation

Centre is used to induce the TBI. VPA, at the dose of 150 mg/kg, is administered via a peripheral intravenous catheter. [6, 32] Details of the model are already published. [33]

TBI Triggers a Genomic and Proteomic Storm

Although genomic responses to injury remain an expanding area of research, the interest in epigenetic mechanisms involved in the pathogenesis of TBI has only recently gained attention. Recent studies demonstrate that TBI results in numerous genomic changes, including modifications in DNA, post-translational changes to histones, and variations at the level of non-coding RNA. [34] These changes are collectively referred to as ‘epigenetics’ and involve modulation of gene expression and phenotype while maintaining the underlying DNA sequence. Of note, these epigenetic changes

following TBI have also been implicated in TBI recovery.[35] Therefore, epigenetic modulation may play a potential role in treatment of TBI. Concurrently, proteomic techniques are being used to analyse TBI pathophysiology in various models. [36] Diffuse TBI has shown to increase the expression of peptides related to neurodegeneration, and decrease the expression of peptides related to antioxidant stress like glutathione S-transferase Mu 3 etc. It has also been shown to increase the expression of potential biomarkers such as neurogranin, fatty acid binding protein etc.[31] Proteomic approaches are well suited for studying changes following TBI, and they may help identify targets for novel treatments.

VPA Decreases the Genomic and Transcriptomic Changes Following TBI

VPA administration attenuates genomic changes following TBI. Using microarray technique, the effects of VPA on brain gene expression in swine subjected to TBI (controlled cortical impact) and haemorrhagic shock (HS) were studied.[37] [38] VPA was found to affect the expression of 370 well-known genes following TBI and HS. The analysis showed that VPA upregulates genetic pathways involved in cell survival, and downregulates pathways involved in apoptosis, necrosis, inflammation and cell death (Table 1). In addition to promoting pro-survival gene expression, VPA upregulates the expression of the neurofilament light (NEFL) gene (Table 2). The NEFL gene encodes the neurofilament light chain protein, a neuronal intermediate filament, that forms the cytoskeleton of axons. Upregulation of the NEFL gene helps in maintaining the functional calibre of neurons. Furthermore, this increase in expression inhibits neuronal apoptosis and promotes axonogenesis. [39]

VPA administration also significantly downregulates genetic pathways involved in inflammatory responses.[38] Following TBI, VPA inhibits the complement system and natural killer inter-cellular communication, dendritic cell maturation, T cell and B cell signalling and pattern recognition receptors in recognition of bacteria and viruses. Several genes are identified that are

involved in these pathways, for eg. CD59, C1QC, and C1QB are involved in complement system pathways; toll-like receptor 3 gene is involved in natural killer inter-cellular communication, dendritic cell maturation and T-call and B-cell signalling.

To further the understanding of mechanisms of VPA in clinically relevant large animal models of TBI, HS, and polytrauma, RNA sequencing was performed to examine the peripheral blood mononuclear cells (PBMCs) of swine. [40] Six hours following administration of VPA, RNA sequencing analysis demonstrated that VPA upregulates genes associated with cell survival, proliferation, and differentiation, while it downregulated genes involved in cell death and inflammation. One day following injuries, repeat analysis confirmed that VPA-induced changes in transcriptome are sustained for at least 24 hours.

Penumbra from injured brains harvested from swine subjected to TBI and HS revealed similar trends. [41] Using RNA sequencing and analysing the results using Ingenuity Pathway Analysis, VPA administration was found to upregulate an inter-connected subnetwork of 257 genes that mediates neurogenesis, neurotransmission, and neuro-regulation (Figure 1). It concurrently downregulated pathways involved in cell death and inflammation. Interestingly, these genomic changes correlated with morphological changes in brains, which showed a decrease in lesion size and edema following

VPA administration.

VPA Influences Proteomic Profiles

The proteome represents the functional expression of genes in the cell.[42] As seen in gene expression profiles following VPA administration, the proteome also undergoes significant changes following VPA treatment.

Proteomic analysis of PBMCs from swine subjected to TBI and HS has shown that VPA results in alterations in the proteome within an hour following administration (Table 3).[43] This rapid induction of proteomic changes suggests that VPA’s mechanisms in trauma may not be limited to increased histone acetylation, as gene transcription is likely to take longer. Several of the altered proteins, like Rho GTPase signalling pathway proteins (Figure 2), are cytoplasmic effector proteins.

This is contradictory to the previous understanding that VPA predominantly affects nuclear histone proteins only. Nevertheless, the altered proteome following VPA administration is found to be implicated in improving survival and mitigating cell death, which is consistent with genomic and transcriptomic studies.

In a human study, similar proteomic changes have been demonstrated following VPA administration. A double-blinded, placebo-controlled, dose-escalation trial (NCT01951560) was performed to gauge the safety and tolerability of ascending doses of VPA in healthy human participants.[25] VPA administration was found to induce proteomic changes in human PBMCs at a dose even lower than the previously tested effective dose in injured swine. [44] The proteomic changes reflected modulation of cell death, cell survival, fatty acid metabolism, neurological disorders, and cellular component organization. Overall, the altered pathways were predominantly related to cellular fate. Interestingly, some of the proteomic alterations suggested a pro-survival role of VPA, while

other alterations were suggestive of cell death and apoptosis. This finding explains how VPA administration can be beneficial in a variety of diseases, like trauma and cancer.

This human study also had some interesting observations regarding VPA-induced acetylation in healthy humans. In contrast to animal studies, the degree of protein acetylation was less profound than expected. Histone H3, in particular, did not exhibit increased acetylation. This may be because the proteome of healthy human PBMCs may not undergo significant changes through histone acetylation.

This variation may also be secondary to the differential effects of VPA in healthy and injured cells, as the proteomic and transcriptomic analyses on PBMCs of injured swine in the preclinical studies have consistently shown a trend towards increased acetylation of histone H3. [40, 43]Although this may be species dependent, this variation in acetylation following VPA administration in healthy humans warrants further studies in trauma patients.

Omics Technologies and Precision Medicine

In animals subjected to TBI, treatment with VPA improves survival, mitigates injury, and facilitates neurological recovery. These findings are clinically relevant since gene expression profiles of HDACs in leukocytes of blunt trauma patients have been demonstrated to be closely related to clinical outcomes.[45]

TBI, however, is highly heterogeneous, and most clinical trials related to TBI have been met with limited success. [15] In addition, according to an in vitro study of human tissue, it was noted that all cells do not respond to HDAC inhibition in a predictable fashion.[46] Due to the heterogeneity of

TBI and VPA’s diverse mechanisms in mitigating injury, it is likely that VPA will be efficacious in some TBI patients, but may have no effect in others.

One of the likely reasons that prior pharmacologic interventions for TBI patients have failed is the enrolment of heterogeneous patient populations in clinical testing. VPA’s promise in treating TBI has resulted in its rapid translation into a successful Phase 1 clinical trial in healthy human subjects.[25]

Phase 2 of clinical testing of VPA in trauma patients with TBI is currently pending. Integral to the successful translation of VPA in Phase II and III trials is making sure that the patient participants that will respond to VPA treatment are enrolled. The goal, therefore, is to develop a point of care (POC) device that can stratify patients based on expected response to VPA treatment, and even stratify them based on injury genotype and phenotype. Advances are already being made in this arena. For example, a POC platelet function device that predicts surgical bleeding risk and guides transfusion in patients on blood thinners has already shown promise. [47] Also, a POC assay that tests for platelet function is being evaluated for guiding platelet transfusion in aspirin users with intracranial hemorrhage.[48]

Using our extensive data repository from various swine studies, [37] [38] [40] [41] [43]and Phase 1 testing of VPA in healthy humans, [44]we are aiming to identify specific omics profiles that can predict response to treatment with VPA. A gene set-pathway analysis is being used to reconstruct VPA’s mechanisms of action that support TBI recovery. The POC device will use buccal swabbing for bedside testing and allow stratifying clinical trial participants by correlating omics data to pharmacogenomic, clinical characteristics, and population variables. The use of such a POC test to screen patients may precede a new age of large clinical trials in the era of personalized medicine.

Conclusions

In conclusion, VPA is a promising agent to treat TBI through HDAC inhibition. Convincing pre-clinical studies have allowed for a rapid translation of VPA into a Phase 1 clinical trial. In mediating this translation, omics technologies have helped elucidate the mechanisms by which VPA improves clinical outcomes in TBI. The knowledge of these mechanisms will be utilized to develop a

POC device for accurate detection of responders and non-responders of VPA in its Phase II of clinical testing.

Acknowledgements

The research included in this review was supported by numerous grants from the U.S. Department of

Defense and the National Institutes of Health to HBA.

Conflict of interest statement

The authors have declared no conflict of interest.

Table 1. Gene Set Enrichment Analysis of Genes Upregulated by VPA.

Biological Process P-value

Nervous system development 5.51E-34

Generation of neurons 1.12E-26

Neurogenesis 1.60E-25

Regulation of nervous system 4.52E-24 development

Regulation of neurogenesis 2.45E-21

Table 2. Genes associated with apoptosis and cell survival with highest fold change following VPA administration.

Expression Gene Symbol Gene Name Function Fold p change

Up CHGB Chromogranin B Hormone 4.72 0.008 (secretogranin 1) activity, exocytosis, and granule secretion

Up NEFL Neurofilament, Structural 4.03 0.01 light polypeptide constituent of cytoskeleton, negative regulation of neuron apoptosis, positive regulation of axonogenesis

Up HTR2A 5- Serotonin 3.53 0.02 hydroxytryptamine receptor (serotonin) signaling and receptor 2A, G activity, protein-coupled synaptic transmission, gap junctions, calcium signaling pathway, neuroactive ligand-receptor interaction, ERK1 and ERK2

Up NR6A1 Nuclear receptor Cell 3.36 <0.0001 subfamily 6, group proliferation, A, member 1 steroid hormone receptor activity, transcription

factor complex

Down ACTC1 Actin, alpha, Cardiac muscle 8.00 0.004 cardiac muscle 1 contraction, apoptosis, hypertrophic cardiomyopathy

Down ASPA Aspartoacylase Aminoacylase 7.78 0.008 activity, amino acid metabolism

Down LOC100513601 Patr class I Immune 7.46 0.004 histocompatibility response, antigen, A-126 antigen alpha chain-like processing and presentation, MHC class 1 protein complex

Down SLCO1A2 Solute carrier Thyroid 4.76 0.005 organic anion hormone transporter family, transmembrane member 1A2 transport, bile acid metabolism, organ regeneration

Table 3. Most significantly elevated proteins within an hour following VPA administration.

Protein name Primary Function Primary Cell P-value Location

Microtubule Actin Cytoskeleton 0.0001 associated depolymerization; monooxygenase inhibits apoptosis

Group specific Vitamin D Cytosol/extracellular 0.0004 component transport/storage; matrix G-actin scavenging

Heat shock protein Protein folding; Extracellular 0.0006 family E member 1 mitochondrial matrix/mitochondria protein biogenesis

Rho guanine Stimulates RhoA Cytosol/plasma 0.0011 nucleotide and dependent membrane exchange factor1 signaling

Ribosomal protein Ribosomal protein Cytosol/extracellular 0.0017 L34 matrix

Figure 1. Molecular network of upregulated genes following VPA treatment in a swine model of TBI and HS. (P = 1xE-27; Fishers exact test).

Figure 2. Condensed visual representation of the Rho GTPase signaling pathway activated by valproic acid (VPA) treatment. Green indicates that the protein/modification was significantly increased (P < 0.05) in the NS D VPA group compared to NS alone at either the postshock or postresuscitation time points. Similarly, red indicates that the protein was decreased in VPA-treated animals at least one time point. Ac [ acetyl group; p [ phosphorylation; ARHGEF1 [ Rho guanine nucleotide exchange factor 1; Rac1 [ Rho family GTPase; ROCK1 [ Rho-associated coiled-coil containing protein kinase 1; IQGAP1 [ IQ motif-containing GTPase activating protein 1; ARP2/3 [ actin-related protein 2/3 complex; MLCK [ myosin light chain kinase; FAK [ focal adhesion kinase; PKN1 [ protein kinase N1; GFAP [ glial fibrillary acidic protein; LIMK [ LIM domain kinase; NADPH [ nicotinamide adenine dinucleotide phosphate; ROS [ reactive oxygen species; NF-kB [ nuclear factor kappa-light-chain-enhancer of activated B cells; ERM [ ezrin/radixin/ moesin protein family; IRS [ insulin receptor substrate; WAVE [ WASP family verprolin-homologous protein family; PI3K [ phosphatidylinositol 3-kinase; N-WASP [ neural Wiskott-Aldrich syndrome protein; SCAR3 [ Drosophila homolog of the mammalian WAVE protein 3; WASP [ Wiskott-Aldrich syndrome protein; CLIP-1 [ CAP-Gly domain containing linker protein 1; PAK [ p21-activated kinase.

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Biography

Dr. Hasan B. Alam is the Norman Thompson Professor of Surgery and Section Head for the Section of General Surgery at the University of Michigan Hospital.

Dr. Alam's clinical interests are in the areas of trauma, emergency general surgery and surgical critical care. His research focuses on hemorrhagic shock, traumatic brain injuries, resuscitation techniques, novel cell preservation strategies, modulation of response to lethal insults, therapeutic hypothermia, and development of new treatments for sepsis. He has published over 300 manuscripts and book chapters and is the holder of 12 patents. He is a member of more than 15 surgical/scientific societies including the American Surgical Association and American Association for the Surgery of Trauma.