Shared PI3K Signaling Abnormalities in Brain Tumors and Epilepsy: PI3K Inhibition in PTEN- Deficient Disorders of the Brain

Shared PI3K signaling abnormalities in brain tumors and epilepsy: PI3K inhibition in PTEN- deficient disorders of the brain

A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of

Doctor of Philosophy in the College of Medicine by

Angela White

B.S. North Carolina Wesleyan College June 2006

Committee Chair: Christina Gross, Ph.D. Lionel M.L. Chow, M.D., Ph.D. Steve C. Danzer, Ph.D. Charles V. Vorhees, Ph.D. Ronald R. Waclaw, M.S., Ph.D.

Abstract

Deficiency in Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN)

(also referred to as MMAC1/TEP1) is known to contribute to multiple diseases of the brain including brain tumors, autism spectrum disorders and epilepsy. PTEN is a dual-specificity phosphatase that negatively regulates the phosphatidylinositol 3-kinase (PI3K) pathway by dephosphorylating phosphatidylinositol (3,4,5)-trisphosphate (PIP3), generating phosphatidylinositol (4,5)-bisphosphate (PIP2). Overactive PI3K signaling increases PIP3 leading to accelerations in cell cycle transition, proliferation and cell size. In this body of work, I assessed PTEN deficiency in the brain in the context of both cancer and neurodevelopmental disorders. In the first studies presented here (Chapters 2 and 3) we assess high grade gliomas.

In Chapter 4, we assess PTEN deficient autism and epilepsy.

Each project has the commonality of utilizing inhibition of the PI3K pathway to counteract the effects of PTEN deficiency but the therapeutic strategies used for each disorder will differ.

For Chapter 2, we found a subset of tumors which have a favorable response to PI3K pathway inhibition, as assessed by proliferation and apoptosis in a novel genetically engineered mouse model which is PTEN deficient. This work seeks to identify the molecular differences in tumors which are reported as responsive to PI3K inhibition as compared to tumors that were not responsive. In Chapter 3, we describe a novel mouse model which represents a subset of pediatric high grade glioma. Due to the driving mutations in the PI3K pathway, including a mutation in PTEN, we assessed both monotherapy PI3K inhibition as well as a monotherapy downstream effector of PI3K (mTOR). Although preliminary, the tumors showed a marked reduction in proliferation.

Finally, in Chapter 4, we assessed a therapeutic approach derived from PTEN-deficient cancers and apply it to PTEN-deficient autism and seizure disorders in a neuron-specific PTEN-

ii deficient mouse model. We reported a significant reduction in several phenotypes including increased protein synthesis, seizures, and certain aspects of macrocephaly.

The broad therapeutic approaches utilized for each PTEN-deficient disorder here are all inhibiting either PI3K or a downstream effector. The specific therapeutic approach is variable and will be better presented in each chapter. Overall, we find potential therapy approaches which will need to be more thoroughly elucidated for their respective disorder.

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Acknowledgements:

First, I would like to thank my current mentor, Nina. From the first time I met with Nina while I was looking into new labs in my mid-PhD transfer, I was warmly welcomed into the lab environment. She spent countless hours helping me learn new techniques, discussing my data, constructively critiquing my work, and also being a support system when I needed it. I would also like to thank my lab members. Not only have they offered help in any way I have ever needed when it comes to the lab and my scientific training, they offered friendship which is treasured. I feel so lucky to have spent time in a lab with such an upbeat atmosphere. Big thanks to Durgesh for all the training (especially the EEG surgery and set up training), the support, and even a few of the bad jokes! I said a few, not all. Lindsay Schroeder-Carter was a smiling and welcoming face each morning when I first joined the Gross Lab. I would not have thrived if I had not received so much training and support from a patient and smiling individual.

Thank you so much! Jeffrey has to be the nicest person I’ve ever met and always there to lend a helping hand or listen! Val, the current lab manager, is also amazing and always there to help the lab in any way! Val is always there to aid and chat about true crime. All of these are needed!

These three lab managers were my life savers more times than I want to think about! Molly and

Andie, thanks for being wonderful students. Your assistance and your enthusiasm are both appreciated! It’s very rewarding to watch you do great things. Lindsay Beasley, thanks for being my early morning chat partner. Amanda and Emma, thank you for being great lab mates! Anna,

Kyle, and Katrina, thanks for being great (almost) lab mates!

Thank you to each member of the Chow Lab (Lionel, Shelly, Ralph, Annmarie, and

Mandi) for your guidance and friendship! Amanda (Mandi) Stang gets a special shout out for all of her technical assistance! Thank you, Lionel, for the mentorship. I also would like to thank the source of funding for my work in the Chow Lab, Sophie’s Angel Run. The Meinhardt family are a source of inspiration and I look forward to running with you again next September!

I would like to thank the committee members of both my thesis committees for all of their feedback and support. My original thesis committee, Lionel Chow, MD, PhD, Ron Waclaw, PhD,

Kathryn A. Wikenheiser-Brokamp, MD, PhD, Nancy Ratner, PhD, and Atsuo T. Sasaki, PhD, thank you all! Importantly, my current thesis committee, thank you for jumping into a project with two different stories: Christina Gross, PhD, Lionel Chow, MD, PhD, Steve Danzer, PhD, Charles

Vorhees, PhD, and Ronald R. Waclaw, MS, PhD. A special thanks to both Lionel and Ron who were on both of my committees!

Thank you to the MDB graduate program for allowing me to receive my training here! I want to say a particular thank you to the faculty members who supported me when I had to move labs. I am full of gratitude for all the kindness and understanding. A HUGE individual thank you to Amanda! Amanda has helped every step of the way-even explaining to me on my first day what a snow emergency is! (I said it then and I stick by my words-this sounds like hell)!

A huge thank you to all of my friends, near and far! Some of my North Carolina friendships have just continued to grow stronger. A special shout out to Theresa and Carrie, my

North Carolina (by way of South Carolina and New York) friends who have gone out of their way to visit me in Cincinnati several times. It was always like having a piece of home here in

Cincinnati. Thank you to all my friends who always rearrange their schedules to see me when I visit NC. The friendships I have made here in Cincinnati are some of the best memories I have of my time here. Diana, thanks for your constant friendship and support. I can’t wait for the world to get normal enough for another Braves vs. Reds game in person! My mom says thanks for inviting me to Thanksgivings too! Sneha, thanks for always being there and listening to me vent!

The dogs and I will miss you at Halloween now that you have moved. A special shout out to several organizations that I am a part of which have brought countless friendships into my life:

Queen City Running Club and Louie’s Legacy. I can’t even count the friendships I have made at

QCRC, but a special shout out to these special runners: Laura, Janet, Nicole, Becki, Ed, Teeny,

Rose, Rick, Leeann, and of course, the fearless leader of QC, Joe. Louie’s has not only brought me the happiness of countless animals and the satisfaction of watching them flourish, it has introduced me to some of the best people ever: Jen, Justy, Cara, Maria, Lisa, and all the West side fosters who I used to spend several hours with at PetSmart every Saturday. I don’t often miss the events but I miss the volunteers!

I also want to specifically acknowledge three very important people who I lost during my

PhD traininng, Jennifer Streicker, Dr. Robert Mullin, and Mabel White. Jen was my direct supervisor and one of the people responsible for hiring me at my previous job. She also holds the titles of hero and friend. I learned so much about science and mice from her. Most importantly, I learned so much about life from her. Take chances and live your life to the fullest- work hard, play hard, and make your impact! Dr. Mullin was a wonderful mentor and friend. I am always grateful for the letters of recommendation that got me here! He was also the first person to hear I got into graduate school and the first person to give me a hug after hearing the news.

Finally, my grandma, Mabel White, thank you for a lifetime of love and support. Whenever times got rough during my PhD, I would think back to our first phone call after I moved to Cincinnati. I will never forget hearing my grandmother say how proud she was of my move and how when she was my age (and by my age she actually meant younger), she wanted to be in medicine but because of her background and the difficulties for women in science in those days, wasn’t able to do so. I always remember that and always remember how lucky I am that I was given different chances.

Thank you to all of my family! When I go home for Christmas, I always feel appreciated by the people who go out of their way to visit with me and I always look forward to those trips. I also am appreciative of the family that has always gone out of the way to text me and check in as a pleasant surprise. To my immediate family, thanks for all the support even though I am pretty sure you all thought I was crazy when I moved away. Thanks to my mom for all the texts

vii and for always sending me random cards and packages to give me a happy surprise. Thanks to my dad for always offering up advice when I needed it and for the trips to Cincinnati to fix things in my house that I should know how to fix. Thank you to my sister who has always done a better job at keeping in touch. An especially big shout out for keeping in close touch this year when my life was especially difficult. Thank you to my nephew for being a great kid and always making me feel like I’m an awesome aunt even though I am so far away. Thank you to my brother-in- law for taking awesome care of those two!

The last thank you is reserved for my dogs, Jonah and Bojangles, who have given me the support and love I have needed any day, all day! …especially Jonah, who moved here with me from North Carolina and probably wishes he had a choice in the matter the first time he experienced snow.

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Abbreviations aCGH Microassay-based Comparative Genomic Hybridization ADHD Attention deficit hyperactivity disorder aHGG Adult high grade glioma AKT Protein kinase B AMPAR α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ASD Autism spectrum disorder BMI-1 B lymphoma Mo-MLV insertion region 1 homolog BP base pairs CamKIIα Alpha-calcium-calmodulin kinase II CBP CREB-binding protein CBR3 Carbonyl reductase CCHMC Cincinnati Children’s Hospital Medical Center CK2 Casein kinase 2 Cnp2 2',3'-Cyclic Nucleotide 3' Phosphodiesterase COSMIC Catalogue of somatic mutations in cancer CNS Central nervous system DG Dentate gyrus DLG1 Discs large MAGUK scaffold protein 1 EGFR-1 Early growth response protein 1 EN2 Engrailed 2 FAK Focal adhesion kinase FCD Focal cortical dysplasia FCP Focus Cancer Panel FXS Fragile X syndrome GBM Glioblastoma multiforme GFAP Glial fibrillary acidic protein GL Granular layer Gli1 Glioma-Associated Oncogene Homolog 1

GSK3β Glycogen synthase kinase 3β GST Glutathione S-transferase H Human

H2O2 Hydrogen peroxide H & E Hematoxylin and eosin HES1 Hairy and enhancer of split-1 HGG High grade glioma ICV Intracerebroventricular ID Intradermally IGF-1 Insulin-like growth factor-I IHC Immunohistochemistry IL2rg IL2 receptor common gamma chain INDELs Insertions/deletions IP Intraperitoneal LOH Loss of heterozygosity LTD Long-term depression MAGI1 Membrane Associated Guanylate Kinase 1 MAGI2 Membrane Associated Guanylate Kinase 2 MAGI3 Membrane Associated Guanylate Kinase 3 MAPK Mitogen-activated protein kinase MAST1 Microtubule Associated Serine/Threonine Kinase 1 MAST2 Microtubule Associated Serine/Threonine Kinase 2 MAST3 Microtubule Associated Serine/Threonine Kinase 3 mGlu1/5 Group 1 metabotropic glutamate MNPs Multi-nucleotide polymorphisms N Number per group NAC N-acetyl cysteine NDRG2 N-myc downstream-regulated gene 2 Nec Necrosis

NEDD2 Neural precursor cell expressed developmentally down-regulated protein 4 NF-1 Neurofibromatosis type I NF-kB Nuclear factor kappa-light-chain-enhancer of activated B NK Severe combined immune deficiency NMDA N-methyl-D-aspartate NPC Neural progenitor cells Nrg-1 Neuregulin 1 NSE Neuron-specific enolase NSG NOD SCID Gamma Olig2 Oligodendrocyte transcription factor 2 P Postnatal PARD3 Par-3 Family Cell Polarity Regulator PBS Phosphate buffered saline PCAF p300/CBP-associated factor PDGFRα Platelet-derived growth factor receptor α PDX Patient-derived xenograft PDZ PSD95, Dlg1, Zo-1 PEST Proline, glutamic acid, serine and threonine PFA Paraformaldehyde PH Pleckstrin homology pHGG Pediatric high grade glioma PHTS PTEN Hamartoma Tumor Syndrome

PIP PI(3)P

PIP2 Phosphatidylinositol (4,5)-bisphosphate

PIP3 Phosphatidylinositol (3,4,5)-trisphosphate

PI3K Phosphatidylinositol 3-kinase PML Polymorphic layer PPAR-γ Peroxisome proliferator-activated receptor γ

PREX2 Phosphatidylinositol-3,4,5-Trisphosphate Dependent Rac Exchange Factor 2 PTEN Phosphatase and Tensin Homolog deleted on Chromosome 10 QKO Gfap-CreER; PtenloxP/loxP; Tp53loxP/loxP; Rb1loxP/loxP; p107-/- RBPJ Recombining binding protein suppressor of hairless Scid Severe combined immune deficiency SF-1 Steroidogenic factor 1 SIRT1 Silent mating type information regulation 2 homolog 1 SLC9A3R1 Solute Carrier Family 9 Member A3 Regulator 1 SNRI Serotonin-norepinephrine reuptake inhibitor SNPs Single nucleotide polymorphisms SPRY2 Sprouty Signaling Antagonist 2 SSRI Selective serotonin reuptake inhibitor SUMO Small ubiquitin-related modifier SVZ Subventricular zone Tp53 Tumor protein 53 TSC Tuberous sclerosis complex WT Wild type WWP2 WW Domain Containing E3 Ubiquitin Protein Ligase 2 XIAO X-linked inhibitor of apoptosis protein

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Table of Contents

Abstract………………………………………………………………... ……….ii

Acknowledgements………………………………………………….... ……..v

Abbreviations………………………………………………….....……….…..ix

Table of Contents………………………………...…………………...……..xiii

Chapter 1: Introduction…………………………………………………………………1

PTEN: from discovery to specific disorders………………………………………2

History of PTEN discovery………………………………………………... ….……2

PTEN Structure…………………………………………………………...... ………3

Effect of Mutations on PTEN Protein Structure…………………………. ………5

PTEN Regulation…………………………………………………………… ………7

PTEN Transcriptional Regulation…………………………………………7

Post-Transcriptional Regulation……………………………….. …………9

Post-Translational Modification………………………………. …………11

PTEN Functions in the Cell…………………………………………….…………13

Function of PTEN in CNS Cell Lines………………………………………….…15

Brain-Specific Genetically Engineered Mouse Models of PTEN Deficiency...18

Therapeutic Interventions in Brain-Specific Mouse Models………………...…23

Therapy in Mouse Models of Brain Tumors……………………… ……24

Therapeutic Approaches in Mouse Models of Neurological

Disorders………………………………………………………………….. 24

Therapeutic Interventions in Clinical Studies………………………...…………27

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Clinical Studies in Cancer…………………………………….. …………27

Clinical Studies in Neurological Disorders…………………….. ………27

Concluding Remarks…………………………………………………………... …28

References……………………………………………………………… ...….……31

Figures and tables……………………… .……………………………………..…59

Dissertation objectives……………………………………………... ………………66

Chapter 2: PI3K inhibition yields varied tumor response in a novel mouse model for high grade glioma…………………………………………………..……67 Abstract……………………………………………………………... …………68

Introduction…………………………………………………………. …………69

Materials and Methods……………………………………………. …………72

Results…………………………………………………………. ………………74

Discussion………………………………………………………...……………80

References………………………………………………………………..……83

Figures and tables…………………………………………………. …………92

Chapter 3: Intracranial patient-derived xenograft from a pediatric glioblastoma retains molecular characteristics and growth pathways…………………...…………108

Abstract…………………………………………………………. ……………109

Introduction……………………………………………………... ……………110

Materials and Methods………………………………………... ……………112

Results………………………………………………………….. ……………114

Discussion…………………………………………………… ………………118

References……………………………………………………... ……………119

Figures and tables………………………………………………...…………124

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Chapter 4: PI3K isoform-selective inhibition in neuron-specific PTEN-deficient mice rescues molecular defects and reduces epilepsy- associated phenotypes…………………………………………………………….132

Abstract………………………………………………………. ………………133

Introduction………………………………………………………………...…134

Materials and Methods………………………………………………... ……137

Results……………………………………………………………….. ………146

Discussion………………………………………………………… …………156

References…………………………………………………………... ………162

Figures and tables……………………………………………………...……173

Chapter 5: Discussion……………………………………………………...………201

The Connection between Cancer and Autism………………… …………202

PTEN Regulation and its Implications in this Research………… ………205

High Grade Gliomas: Implications of Dissertation Research……... ……206

Potential Utility of our Findings for High Grade Glioma Research…….. 208

QKO Mouse Model Research………………………………….. …208

Pediatric HGG Modeling and Future Research……………. ……211

Limitations and Potential Implications of High Grade Glioma

Research………………………………………………………………………212

Implications of p110β Inhibition in PTEN-deficient Autism……………... 214

Outlooks of PTEN Deficiency Research and Potential Therapy.214

Assessing Potential Disease Modifying Effects and Underlying Mechanisms of p110β Inhibition……………………………….. …215

The Connection of PI3K Isoforms and Neurological Disorders...219

Research Considerations and Limitations for Chapter 4………. 223

Concluding Remarks………………………………………….….…225

References………………………………………………………… ..226

Tables………………………………………………………………...244

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Lists of Figures and Tables

Chapter 1: Introduction

Figure 1: The Domains of PTEN ……………………………………….. …………..59

Figure 2: Mutations of the PTEN Gene…………………………………… ………..60

Figure 3: PTEN is the main negative regulator in the PI3K pathway.…...... …..61

Table 1: Proteins that bind to PIP3 through a PH domain interaction…. ………..62

Table 2: Ion Channels and Transporters Regulated by PIP2……………………..64

Table 3: Cancer clinical trials which utilize inhibition in the PI3K pathway…...….65

Chapter 2: PI3K inhibition yields varied tumor response in a novel mouse model for high grade glioma Figure 1: QKO tumors closely mimic human HGGs……………………………….92

Figure 2: QKO tumors respond similarly to standards of care……………………94

Figure 3: Short-term treatment reduces PI3K signaling in QKO tumors…………96

Figure 4: Tumor response is variable in QKO tumors subjected to a combination of PI3K and mTOR inhibition……………………………………...... 98

Figure 5: KI67 positivity correlates with GSX2 and negatively correlates with Sox9 in immunohistochemical staining…………………………… 99

Figure 6: Cleaved caspase-3 negatively correlates with Sox10………...……... 100

Supplemental Figure 1: Tumors at 6 weeks post Tamoxifen……………………103

Supplemental Figure 2: Tumor stem cell properties of HGGs……...... ………102

Supplemental Table 1: Antibodies used for all IHC experiments…….…………103

Table 1: Completed clinical trials for solid tumors which utilize PI3K inhibition as well as the reported response………………………….. ……………104

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Table 2: IHC markers for the identification of progenitor cells as well as more differentiated cells within QKO tumors………………...……………105

Table 3: IHC markers which correlate to KI67………………………….…………106

Table 4: IHC markers which correlate to cleaved Caspase-3……………………107

Chapter 3: Intracranial patient-derived xenograft from a pediatric glioblastoma retains molecular characteristics and growth pathways

Figure 1: Conservation of H3F3A G34R mutation in patient-derived tumors.…………………………………………………………………………………124

Figure 2: Histology of pGBM PDX………………………………………………….125

Figure 3: PI3K Pathway and Inhibition……………………………………………..127

Figure 4: Response to PI3K pathway inhibition…………………………….……..128

Table 1: Antibodies utilized for immunohistochemical staining…………...……..129

Table 2: Driver mutations……………………………………………………..……..130

Table 3. Conservation of driver mutations in PDX tumors………………...……..131

Chapter 4: PI3K isoform-selective inhibition in neuron-specific PTEN-deficient mice rescues molecular defects and reduces epilepsy-associated phenotypes

Figure 1: Elevated AKT phosphorylation in PTEN-deficient neuronal cultures is reduced to control levels with selective p110β inhibition…………… 173

Figure 2: Selective p110β inhibition normalizes aberrant cell signaling and protein synthesis associated with PTEN deficiency in acute cortical and hippocampal mouse brain slices………...……………………………175

Figure 3: Administration of GSK6A reduces elevated AKT phosphorylation and increases survival in Pten; CamKdel mice…….. …………178

Figure 4: GSK6A improves nest building behavior and reduces aberrant

PI3K signaling in Pten; CamKdel mice………………..……………………………180

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Figure 5: GSK6A administration reduces seizure frequency in Pten;

CamKdel mice…………………………………………………….. ………...... …….182

Figure 6: Chronic GSK6A administration reduces enhanced AKT phosphorylation and normalizes brain weight in Pten; Gfapdel mice…. ……….184

Figure 7: Intraperitoneal GSK6A administration reduces cortical enlargement and hippocampal soma size to control levels in Pten; Gfapdel mice…………... 186

Figure S1: mRNA and protein expression of p110 isoforms remain unchanged in primary neuronal cultures after Pten siRNA transfection……… ..189

Figure S2: Mechanisms of p110β regulation may differ in PTEN-deficient cancer cells and PTEN-deficient primary neurons……………………………….. 191

Figure S3: Protein expression of p110 isoforms remains unchanged in

Pten; CamKdel mice………………………………………………………………… 193

Figure S4: Oral GSK6A administration at 5 mg/kg in peanut butter reduces elevated AKT phosphorylation in Pten; CamKdel mice………. ……….194

Figure S5: Pten; CamKdel mice have increased S6 Kinase phosphorylation, but not S6 phosphorylation in cortex and hippocampus…………………….. …..195

Figure S6: GSK6A administration may reduce gamma EEG power and frequency of EEG spikes and spike trains in Pten; CamKdel mice………. …….197

Table 1: Isoform-selectivity of GSK2702926A, IC87114, and BYL-719…….…199

Table 2: Mortality throughout EEG studies in Pten; CamKdel mice………….…200

Chapter 5: Discussion

Table 1: Shared pathways and cellular processes dysregulated in cancer and autism…………………………………………. …………………………………244

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Table 2: Hypothetical experimental design for mouse study which targets both differentiated and stem cells………………….. ………………………………245

Table 3: Hypothetical experimental design for patient-derived xenograft study………………………………………………………... …………………………246

Table 4: Current therapeutic approaches used for the treatment of autism symptoms…………………………………………………..…………………247

Chapter 1: Introduction

PTEN: From Discovery to Specific Disorders

Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN) (also referred to as MMAC1/TEP1) is a dual phosphatase with both protein and lipid phosphatase activities

(Abdulkareem & Blair, 2013). PTEN plays important roles in many cellular functions such as growth and survival (Smith & Briggs, 2016). It is associated with multiple cellular overgrowth and migration disorders, including cancer and autism (Eng, 1993). Here, we examine PTEN from a historical perspective and discuss its known structural components and function. Moreover, we describe PTEN-deficient in vitro models and their implications on cellular architecture, as well as in vivo mouse models, which target specific cell populations of the central nervous system

(CNS), and the implications of temporal, spatial, and concentration of PTEN deficiency, specifically in the CNS. Additionally, we highlight specific PTEN mutations, the consequence of these mutations to the protein, and the corresponding disorder associated with the mutation in the context of both brain tumors, as well as autism.

History of PTEN Discovery

The PTEN gene was originally discovered in a comprehensive set of experiments aimed at identifying consistently mutated genes in cancers. Chromosome 10q23 was isolated as a common loss of heterozygosity (LOH) location. Eventual sequence analysis identified the deletion of 180 base pairs (bp) of exonic sequence and the splice donor site from this 225-bp exon (J. Li et al., 1997; Steck et al., 1997). A thorough discussion of the structure of PTEN occurs in the subsequent section.

Shortly after its discovery in cancer, germline mutations were identified in a set of autosomal dominant syndromes, referred to as PTEN Hamartoma Tumor Syndrome (PHTS)

(Nelen et al., 1996). These syndromes are hallmarked by benign, tumor-like formations

(hamartomas), neurodevelopmental disorders, and a predisposition to multiple cancers

(including but not exclusive to thyroid, breast, endometrial and kidney) (Hobert & Eng, 2009).

The link between PTEN mutations and autism was discovered after there was an observation that families with the PTEN-linked disorder known as Cowden Syndrome have a higher incidence of autism. To present day, this observation has led to a known association between PTEN mutations and individuals with both autism and macrocephaly (Buxbaum et al.,

2007).

The original description of PTEN led to more than 130 papers published within two years, assessing the structure, regulation, and function. First, we will assess the structure of

PTEN and the importance of each domain.

PTEN Structure

PTEN consists of four domains and a binding motif: the N-terminal domain, the phosphatase domain, the C2 domain, and the C-terminal tail, which contains a PDZ binding motif, often wrongly referred to as the PDZ domain. PDZ domains (named for the initial proteins that were found to have these domains: postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 protein (zo-1)) are often present in scaffolding proteins, and enable the formation of protein complexes (Jerde, 2015). A simplified illustration of the domains is shown in Figure 1.

The N-terminus of PTEN contains both independent and overlapping nuclear localization signals and PI(3)P (PIP) binding motifs that regulate PTEN nuclear localization, as well as its catalytic activity (Campbell, Liu, & Ross, 2003). Mutations of the N-terminal portion of PTEN have been shown to alter PTEN’s ability to bind to the plasma membrane (Nguyen et al., 2015).

Interestingly, Gil et al found, via the Catalogue Of Somatic Mutations In Cancer (COSMIC)

3 database, that over 15% of known tumor mutations are found in residues 1-40 of the N-terminal

(Gil et al., 2015).

The phosphatase domain, which is responsible for the enzymatic activity of PTEN, consists of a β sheet of five layers. This β sheet is compacted by α helices on two sides. Within the active site of the phosphatase domain, a loop (known as the p loop) occurs at residues 123 through 130. This p loop, when first assessed, was quickly considered similar to active sites found in protein tyrosine phosphatases and dual specificity protein phosphatases (J. O. Lee et al., 1999). The pocket is given a wider conformation due to a “TI” loop which is stabilized by contacts with other portions of the phosphatase domain. This larger conformation is needed to accommodate the large size and binding to the PI(3,4,5)P3 (PIP3) substrate (Yuvaniyama,

Denu, Dixon, & Saper, 1996).

The C2 domain binds to phospholipid membranes and contains sequences which are responsible for attachment in both protein interactions and cell localization (Raftopoulou,

Etienne-Manneville, Self, Nicholls, & Hall, 2004; Rizo & Sudhof, 1998). The PTEN C2 domain is composed of two anti-parallel β-sheets (Georgescu et al., 2000). This domain is able to bind to lipids after conformational changes, which leaves the region open, in the carbonyl reductase

(CBR3) region and C2 region which connects the two β-sheets (J. O. Lee et al., 1999).

Interestingly, many C2 domains function by cohesion to Ca2+ via acidic residues. However,

PTEN’s C2 domain contains basic residues and binds directly to membranes in a manner independent of Ca2+ (Essen, Perisic, Cheung, Katan, & Williams, 1996; Georgescu et al., 2000).

The C-terminal tail of PTEN is comprised of residues 354 to 403. Experimental deletion of the tail has shown that it does not contribute to the enzymatic functions of PTEN (Georgescu,

Kirsch, Akagi, Shishido, & Hanafusa, 1999). Although no clear enzymatic function is known, the

C-terminal tail undergoes conformational changes as a result of phosphorylation (at sites Ser-

370, Ser-380, Thr-382, Thr-383, and Ser-385) (Torres & Pulido, 2001). When phosphorylated,

PTEN remains in a closed conformation and is considered highly stable. By contrast, PTEN is very unstable (reported as a fourfold decrease in half-life) in the unphosphorylated and open conformation (Vazquez, Ramaswamy, Nakamura, & Sellers, 2000). The open conformation makes the protein enzymatically active. However, this conformation also leaves proline (P), glutamic acid (E), serine (S), and threonine (T) (PEST) sequences at residues 350 to 375 and

379 to 386 exposed (Vazquez et al., 2000). These sequences aid in targeting proteins for proteolytic degradation (Rogers, Wells, & Rechsteiner, 1986).

The last three amino acids of PTEN comprise the PDZ-binding motif and allow for PTEN to adhere to PDZ-binding proteins. This cohesion yields recruitment to the plasma membrane

(Valiente et al., 2005). Proteins that interact with the PDZ-binding motif include Discs Large

MAGUK Scaffold Protein 1 (DLG1), Membrane Associated Guanylate Kinase 1, 2, and 3

(MAGI1, MAGI2, MAGI3), Microtubule Associated Serine/Threonine Kinase 1, 2 and 3 (MAST1,

MAST2 and MAST3), Par-3 Family Cell Polarity Regulator (PARD3), Phosphatidylinositol-3,4,5-

Trisphosphate Dependent Rac Exchange Factor 2 (PREX2), and Solute Carrier Family 9

Member A3 Regulator 1 (SLC9A3R1) (Hopkins, Hodakoski, Barrows, Mense, & Parsons, 2014).

Effect of Mutations on PTEN Protein Structure

With a strongly established association of PTEN dysregulation with cancer, PHTS, and autism, it is important to understand the role of specific PTEN mutations and the resulting phenotypes. To assess the genotype to phenotype relationship seen in PTEN-associated autism and PHTS, Spinelli et al expressed PTEN with autism-associated or PHTS-associated mutations, or wild type (WT) PTEN in a PTEN-null cancer cell line (U87MG). Seven autism- associated mutations were assessed and each of the resulting autism-associated cell lines displayed decreased downstream PI3K signaling in a manner equivalent to WT PTEN (Spinelli,

Black, Berg, Eickholt, & Leslie, 2015). It is notable that these autism-associated proteins are known to be highly unstable and would have been expected to be present in very low quantities in normal physiological conditions, making these results difficult to interpret. Five PHTS- associated mutations yielded no decrease in PI3K signaling and suggest these mutations yield a loss of stability in normal biological conditions. Collectively, this work suggested that there is an association between the gene mutation, the cellular phenotypes, and the resulting disorder

(Spinelli et al., 2015).

A subset of autism-associated mutations are unable to localize to the nucleus (Fricano-

Kugler et al., 2018). This work also demonstrated that forced nuclear localization can partially restore the aberrant phenotype of increased soma size suggesting nuclear PTEN is partially responsible for abnormal cellular phenotypes. This partial rescue is attributed to PIP3 found within the nuclear matrix (Lindsay et al., 2006). Interestingly, a CRISPR/Cas9 mouse model which exclusively deletes nuclear PTEN has reported microcephaly and decreased soma size, indicating that the context of PTEN deficiency within the cell remains to be fully elucidated

(Igarashi et al., 2018).

In a recent study, germline mutations associated with cancer were compared to mutations associated with autism (Smith, Thacker, Jaini, & Eng, 2019) and cancer-associated mutations appeared to have the most alterations in PTEN structure. These structural changes are collectively attributed to both the overall stability of the protein, as well as the structural dynamics. Interestingly, mutations known to be associated with both cancer and autism had the second most alterations in structure (Smith et al., 2019).

Figure 2 is a representation of the mutations that have been studied in order to assess the genotype to phenotype relationship. Mutations for each disorder are found throughout the gene. There does appear to be a correlation between the disorder and the resulting protein. As previously mentioned, cancer mutations generate a change to the structure of PTEN (Leslie et

6 al., 2001; Smith et al., 2019). Mutations associated with cancer which yield PTEN protein that retains partial activity result in mild phenotypes. Loss of protein, in the context of cancer, generates intermediate phenotypes and stable proteins which are rendered inactive produce the most severe phenotypes (Leslie et al., 2001; Leslie & Longy, 2016; Tibarewal et al., 2012).

PTEN Regulation

Most original assessments of PTEN-deficient function were associated with mutations in the gene; however, it is now known that PTEN deficiency occurs on many different levels, ranging from gene mutation through post-translational modifications. Here we discuss additional ways in which PTEN is regulated, which are not caused by a loss-of function mutation. It is worth noting that much of what is known about the regulation of PTEN has been studied in the context of cancer and is therefore what will be included here. Because the physiological differences in cancer cells and neurons, it is not well understood how much of what is known about PTEN regulation will eventually prove pertinent in the (non-cancerous) brain.

PTEN Transcriptional Regulation In addition to gene mutations, PTEN is regulated in a multitude of ways. Beginning at the level of gene expression, PTEN is positively and directly regulated by the transcription factor early growth response protein 1 (EGFR-1), the nuclear receptor peroxisome proliferator- activated receptor γ (PPAR-γ), and the tumor suppressor tumor protein 53 (Tp53; also known as p53) by binding to the promotor region of PTEN (Patel et al., 2001; Stambolic et al., 2001).

Additionally, it has been shown in HeLa cells and primary mouse embryonic fibroblasts (PMEFs) that human Sprouty Signaling Antagonist 2 (SPRY2), which is a negative feedback regulator of multiple receptor tyrosine kinases, directly regulates PTEN expression and inhibitory

7 phosphorylation of PTEN is decreased in cells expressing SPRY2 (Edwin, Singh, Endersby,

Baker, & Patel, 2006).

Notch signaling is able to either positively or negatively regulate PTEN. Notch signaling regulates the expression of hairy and enhancer of split-1 (HES1) which yields a downregulation of PTEN (Palomero et al., 2007). Conversely, Notch signaling inhibits recombination signal- binding protein Jκ (RBPJ) yielding an upregulation of PTEN (Chappell et al., 2005; Whelan,

Forbes, & Bertrand, 2007). Notch signaling, including the activation of downstream effectors, are considered context-dependent.

It has been shown in a panel of human tumor samples that the transcription factor c-Jun has an inverse proportion of protein levels in comparison to PTEN. Additionally, Hettinger et al found that overexpression of c-Jun led to increased cell survival, elevated phospho-Akt (pAkt) levels, and a diminished level of PTEN (Hettinger et al., 2007). The mechanism is not fully understood, but it is hypothesized that c-Jun binds to the promotor of PTEN in a manner similar to what has been noted in c-Jun and suppressing p53 interactions (Hettinger et al., 2007). It will be discussed more thoroughly throughout this work that pAkt is found downstream of PTEN and is often used as a readout to assess PTEN activity.

A protein generally classified as an antiapoptotic transcription activator, nuclear factor kappa-light-chain-enhancer of activated B (NF-kB), was found to negatively regulate PTEN

(Vasudevan, Gurumurthy, & Rangnekar, 2004). This finding introduces a feedback loop in which

PTEN regulates NF-kB concentrations through its regulation of Akt. In turn, Akt activates the

NF-κB protein p65 (Bai, Ueno, & Vogt, 2009).

Additional negative regulators of PTEN have been found in specific context (Bermudez

Brito, Goulielmaki, & Papakonstanti, 2015). An example is polycomb group protein B lymphoma

Mo-MLV insertion region 1 homolog (Bmi-1). Overexpression of Bmi-1 has been found in tumors

8 with a high propensity of metastasis. The regulation of PTEN in these tumors is hypothesized to be a contributor of the epithelial to mesenchymal transition that hallmarks metastasis, although the mechanism is not well understood (Meng et al., 2012; L. B. Song et al., 2009).

Another interesting context-specific regulation of PTEN occurs in ovarian cancer. In many instances, E-cadherin functions as a tumor suppressor, but in ovarian cancer E-cadherin downregulates early growth response gene 1 (Egr1) which directly regulates PTEN (Lau,

Klausen, & Leung, 2011).

Epigenetic silencing has also been implicated in PTEN downregulation in many forms of cancer. It has not been as well described in the context of other disorders such as autism.

Hypermethylated CpG islands in the promotor region of PTEN have been documented in melanoma, gastric, colorectal, thyroid, and breast cancers (Alvarez-Nunez et al., 2006; Goel et al., 2004; Kang, Lee, & Kim, 2002; Khan et al., 2004; Mirmohammadsadegh et al., 2006). To our knowledge, epigenetic silencing of PTEN has not been implicated in brain disorders other than brain tumors (O'Brien, Kreso, & Jamieson, 2010).

Post-Transcriptional Regulation A wide array of microRNAs (miRNAs) are known to regulate PTEN. MiRNAs are small, non-coding RNAs which regulate gene expression by degrading or suppressing the translation of their target RNA (Bartel, 2018). Of importance, two miRNAs, miR-21 and miR-429, often associated with tumors have also been shown to directly target PTEN. In triple-negative breast cancer, colorectal cancer, ovarian cancer, and non-small cell lung cancer, miR-21 upregulation and its control of PTEN was shown to increase tumor invasiveness (Dong et al., 2014; Z. L. Liu,

Wang, Liu, & Wang, 2013; Lou, Yang, Wang, Cui, & Huang, 2010; Xiong et al., 2014). MiR-21 is found to be upregulated in the brain in the context of oxygen-glucose deprivation, but it is unknown if it regulates PTEN (Di et al., 2014). MiR-429 is known to function in a similar manner in human non-small cell lung tumors (Lang et al., 2014).

Additionally, miR-214, miR130a, and miR-93 bind to the 3’-UTR of PTEN ultimately leading to a decrease in PTEN expression. Subsequently, this control leads to increased cell survival and chemotherapy resistance in human ovarian cancer (Fu, Tian, Zhang, Chen, & Hao,

2012; H. Yang et al., 2008; Z. Yang et al., 2019). In glioblastoma, miR-221 alters cellular proliferation and chemotherapy resistance by binding to the 3’-UTR of PTEN. This can be rescued in tissue cultures by the overexpression of PTEN which lacks a 3’-UTR (Q. Xie, Yan,

Huang, Zhong, & Huang, 2014). A similar mechanism of action has been reported in breast cancer with miR-29b, miR-301, and miR-301a (Ma et al., 2014; W. Shi et al., 2011; C. Wang,

Bian, Wei, & Zhang, 2011).

MiR-26a has been shown to negatively regulate PTEN in both a glioma mouse model and in human lung cancer (Huse et al., 2009; B. Liu et al., 2012). Additionally, miR-153 increases proliferation in prostate cancer cells through its direct regulation of PTEN (Z. Wu, He,

He, & Mao, 2013). MiR-494, miR-92a, miR-32, and miR-103 all regulate PTEN levels in colorectal cancers (Geng et al., 2014; Ke, Wei, Yeh, Chen, & Cheng, 2015; H. B. Sun et al.,

2014; W. Wu et al., 2013). MiR-153 also functions to regulate PTEN in human cell cultures of prostate tumor cells (Z. Wu et al., 2013). Recent work in osteosarcoma has identified miR-200 as a promotor of PTEN which yields an upregulation in PD-L1-mediated immunosuppression (Z.

Liu et al., 2020).

Due to the contributions of PTEN to tumorigenesis, much of what is known about post- transcriptional regulation of PTEN has been studied in the context of tumors. It is difficult to understand how miRNAs control PTEN regulation in other diseases that are known to have aberrant PTEN levels. A recent study assessed PTEN regulation in brain ischemia. This work demonstrated miR-21 negatively regulates PTEN, consequently reducing neural apoptosis in an ischemic mouse model (J. Li, Lv, & Che, 2020). MiR-21 is also known to directly target PTEN in the context of axon regeneration in traumatic brain injury (Han et al., 2014; H. Z. Liu et al.,

2014). In this work, overexpression of miR-21 in vitro reduced neuronal sensitivity to apoptosis

(Han et al., 2014).

The number of microRNAs that are found to regulate PTEN is continually increasing.

The understanding of how PTEN is regulated in this capacity is far from complete.

It is known that many of these miRNA themselves are subject to control, further regulating the concentration of PTEN. A pseudogene of PTEN, known as PTENpg1 (also known as PTENp1, PTEN2 and PTENΨ1) functions as a “decoy gene” and ultimately has retained multiple sequences identical to PTEN and therefore actively competes with PTEN miRNAs

(Poliseno et al., 2010). Further assessment of PTENpg1 has shown that the gene encodes for three dominant antisense RNAs (unspliced, α, and β). These RNAs have been shown to epigenetically regulate PTEN (Johnsson et al., 2013).

Post-Translational Modification

As previously described, the PTEN protein has four domains and the PDZ binding motif, each of which contributes an important role in protein function. Post-translational modification of any of these domains can significantly modify PTEN protein interactions, localization, and stability.

PTEN phosphorylation is prevalent in the C-terminal tail of the protein. Ser362, Thr366,

Ser370, Ser380, Thr382, Thr383, and Ser385 are often phosphorylated by casein kinase 2

(CK2) (Al-Khouri, Ma, Togo, Williams, & Mustelin, 2005; Torres & Pulido, 2001; Vazquez et al.,

2000; Xu, Yao, Jiang, Lu, & Dai, 2010). Glycogen synthase kinase 3β (GSK3β) is responsible for the phosphorylation of sites Ser362 and Thr366 (Al-Khouri et al., 2005). As described above, increases in phosphorylation of the C-terminal tail of PTEN yields a closed conformation of the protein. Although this conformation creates a more stable protein, its catalytic activity is hindered.

The C2 domain has multiple phosphorylation sites and effectors. RhoA-associated kinase phosphorylates Ser229, Thr232, Thr319, and Thr321; and RAK phosphorylates Tyr336

(Z. Li et al., 2005; Yim et al., 2009). Focal adhesion kinase (FAK) phosphorylates PTEN on

Tyr336 and is implicated in PTEN membrane association (Tzenaki, Aivaliotis, & Papakonstanti,

2015).

Reciprocally, the dephosphorylating of PTEN is equally important for the purpose of protein stability and binding. It is well documented that PTEN has a lipid phosphatase function, as well as the ability to auto-dephosphorylate (Raftopoulou et al., 2004; Tibarewal et al., 2012).

In addition to auto-dephosphorylation, N-myc downstream-regulated gene 2 (NDRG2) binds to and recruits additional protein complexes to PTEN. This correlates with an increase in PTEN dephosphorylation (Nakahata et al., 2014).

Only a few acetylation events have been reported for PTEN. The histone acetyltransferase, p300/CBP-associated factor (PCAF) downregulates PTEN catalytic activity by interacting with amino acids 186-202 (Okumura et al., 2006). CREB-binding protein (CBP) also acetylates PTEN in prostate cancer; however, the mechanism is not well understood (Ding et al., 2014). Silent mating type information regulation 2 homolog 1 (SIRT1) deacetylates PTEN, yielding an increase in phosphatase activity (Chae & Broxmeyer, 2011).

Hydrogen peroxide (H2O2) oxidizes PTEN by forming a disulfide bond between Cysteine

71 and 124 yielding a decrease in PTEN activity (Cho et al., 2004; Leslie et al., 2003).

Downstream effectors of PTEN, including Akt, have been shown to be significantly increased in models which lack peroxidase, the enzyme responsible for the production of H2O2 (Cao et al.,

2009).

Small ubiquitin-related modifier (SUMO) proteins SUMO1 and SUMO2 are conjugated to

PTEN in vitro. It has been demonstrated, in human and mouse cancer cell cultures, that

12 sumoylation of PTEN increases membrane association of PTEN by inhibiting intramolecular reactions of PTEN (Gonzalez-Santamaria et al., 2012).

Several ubiquitination ligases have been verified in the degradation of PTEN: X-linked inhibitor of apoptosis protein (XIAP), WW Domain Containing E3 Ubiquitin Protein Ligase 2

(WWP2), and neural precursor cell expressed developmentally down-regulated protein

4 (NEDD4) (Eide et al., 2013; Fouladkou et al., 2008; Maddika et al., 2011; Van Themsche,

Leblanc, Parent, & Asselin, 2009). After ubiquitination but prior to degradation, there are deubiquitination enzymes which are able to remove these degradation markers, further regulating PTEN (M. S. Song et al., 2008).

PTEN Functions in the Cell

After its identification as a potential tumor suppressor with key structural features similar to protein tyrosine phosphatases, Myers et al overexpressed PTEN in Escherichia coli as a glutathione S-transferase (GST) fusion protein (Myers et al., 1997). This work concluded that

PTEN was able to dephosphorylate serine, threonine, and tyrosine residues, establishing its function as a dual-specificity protein phosphatase. Overexpression of PTEN in both PTEN- deficient and PTEN wild type cancer cell lines yielded an increase G1 cell cycle arrest (Davies et al., 1998; Weng et al., 1999).

Around the same time as protein phosphatase function of PTEN was being discovered, multiple labs were beginning to understand the most well-known function of PTEN, its lipid phosphatase activity. It was concluded PTEN exerts its lipid phosphatase activity to negatively regulate the phosphatidylinositol 3-kinase (PI3K) pathway by dephosphorylating phosphatidylinositol (3,4,5)-trisphosphate (PIP3), generating phosphatidylinositol (4,5)- bisphosphate (PIP2) (Figure 1) (Maehama & Dixon, 1998; X. Wu, Senechal, Neshat, Whang, &

Sawyers, 1998). PIP3 is an intracellular second messenger which is generated in response to multiple cellular stimuli, including growth factors and hormones (Cantrell, 2001; Toker, 2000;

Wymann & Pirola, 1998). PIP3 binds to and regulates proteins which have a pleckstrin homology (PH) domain (Table 1, modified from Ayyadevara, et al) (Ayyadevara et al., 2016;

Lemmon & Ferguson, 2000; Vanhaesebroeck & Waterfield, 1999).

There are multiple proteins that have been shown to bind to PIP3 (Ayyadevara et al.,

2016). One of the most well documented and studied proteins is protein kinase B (Akt). The finding that PTEN regulates PIP3 and subsequently Akt introduced the concept that PTEN functions as a negative regulator of PI3K/Akt/mTOR pathway (Maehama & Dixon, 1998).

Subsequent work in numerous cell lines with targeted deletion of Pten concluded the relative levels of PIP3 were significantly elevated when compared to control cell lines (Stambolic et al.,

1998; H. Sun et al., 1999). Human tumor cell cultures with known PTEN depletion have an increase in PIP3 as compared to human tumor cell cultures in which PTEN expression is not altered (Taylor et al., 2000).

Apart from its role in PI3K signal transduction, PTEN regulates synaptic localization.

PTEN plays a vital role in the regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) function and plasticity through its regulation of PIP3 (Arendt et al.,

2010).

A major segment of the work to assess PTEN function(s) has focused on lipid phosphatase activity on PIP3 negatively regulating the PI3K pathway; however, PTEN has multiple alternative functions. In 2003, Freeman et al found that PTEN binds to p53 and alters its transcription and protein levels (Freeman et al., 2003). PTEN and PI3K inhibitors were also found to upregulate p53 and alter angiogenesis in human tumor cell cultures (J. D. Su, Mayo,

Donner, & Durden, 2003). P53 is the only protein that is aberrantly regulated more frequently in

14 cancer than PTEN. Therefore, this regulation has proven pivotal in cancer therapeutics

(Issaeva, 2019).

An additional contribution that PTEN may have to tumorigenesis is the suppression of microspherule protein 58 (MSP58) through mechanisms not fully understood. MSP58 is documented to aid in cellular proliferation (Hsu et al., 2014; Okumura, Zhao, Depinho, Furnari,

& Cavenee, 2005).

The discussion of PTEN function thus far has been focused on its role in the cytoplasm of the cell; however, as mentioned above, PTEN is able to reside within the nucleus and has additional function within this context. Nuclear PTEN modulates the activity of the PIP-binding orphan receptors steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1) (Krylova et al., 2005; Ortlund et al., 2005). Additionally, nuclear PTEN downregulates cyclin D1 and prevents phosphorylation of mitogen-activated protein kinase (MAPK) (Chung & Eng, 2005).

This work also showed cancerous cells have a lower quantity of nuclear PTEN than non- cancerous cells, supporting an argument that nuclear PTEN is important for normal cellular function. Mutations frequently associated with autism phenotypes promote neuronal hypertrophy and forced nuclear localization is sufficient to rescue increased soma size (Fricano-

Kugler et al., 2018).

Much of the focus of PTEN function emphasizes the abundance of PIP3 that is a result of PTEN deficiency; however, PIP2 has important cellular functions and thus its depletion can also contribute to disease. Many proteins which are involved in endocytosis and exocytosis are recruited and bound to the cell membrane due to their binding to PIP2 (Huang, 2007). Table 2 lists ion channels and transporters which are regulated by PIP2 (Park, Park, & Suh, 2017; Suh

& Hille, 2005, 2007, 2008; Suh, Kim, Falkenburger, & Hille, 2012).

A lot of our understanding about the multiple functions of PTEN has come from cell culture work and has been translated into animal models. Next, we discuss PTEN deficiency in the context of cell culture followed by animal models.

Function of PTEN in CNS Cell Lines

PTEN is known to suppress cell proliferation and growth in brain tumors and the CNS.

Much of this work has been assessed in the context of specific disorders. Included here are the implications to PTEN deficiency in cultured brain tumor models and neurons. It is important to note that while brain tumor cells are dividing, PTEN deficiency in mature neurons is unique as compared to many other cells, because they no longer divide (Harkany et al., 2004). Here, we will discuss the effects of PTEN deficiencies on both cell types in the CNS, dividing brain tumor cells and differentiated neurons.

Brain Tumor PTEN-deficient Cell Cultures

Many studies which examine PTEN-deficient cell cultures in order to assess brain tumor biology employ established glioma cell lines U87MG, A172, and T98G. These tumor cell lines were established from human tumors. It has been shown in a multitude of studies utilizing these and other brain tumor cell lines that an array of both PI3K and mTOR inhibitors are able to decrease PI3K signaling and reduce cell growth and proliferation (Daniele et al., 2015; Mallon et al., 2011; M. Y. Wang et al., 2006; Zhan et al., 2017; Zhao, Chen, & Liang, 2019). It is worth noting that these cell lines have lost some of the initial characteristics after years of countless cell passages (Kiseleva, Kartashev, Vartanyan, Pinevich, & Samoilovich, 2016). Although T98G and A172 have retained some (but not all) of the characteristics of the donating tumor, U87MG has a very different DNA profile as compared to the donating tumor (Allen, Bjerke, Edlund,

Nelander, & Westermark, 2016). Patient-derived glioma models are becoming more prevalent and are arguably a more accurate depiction of tumor biology; however, each tumor must be

16 genetically profiled upon initiation of the culture which is an additional time and monetary restraint (da Hora, Schweiger, Wurdinger, & Tannous, 2019).

PTEN-deficient Neuronal Cultures

In humans, PTEN deficiency in the brain is known to be associated with macrocephaly, neurodevelopmental delays, and autism (Buxbaum et al., 2007). As previously mentioned, the link between autism with macrocephaly and PTEN mutations is so pronounced that genetic testing for PTEN mutations is a recommendation for individuals with macrocephaly accompanied with autism (Buxbaum et al., 2007). Organoid cultures have utility in assessment of autism, as they are an in vitro representation of some of the characteristics of a developing brain. Organoids show that PTEN deficiency extends cellular proliferation and delays in neuronal differentiation (Y. Li et al., 2017). PTEN depletion also increases progenitor cell renewal in neural stem cells (Zheng et al., 2008).

PTEN deficiency has been studied in neuronal cultures in order to assess spinal cord injury and PTEN depletion as a potential therapeutic strategy. Chemical depletion of PTEN levels significantly increases neurite growth and size (Sopasakis et al., 2010). Additionally, primary neurons subjected to short hairpin RNA silencing of PTEN exhibit neurite elongation, as well as synaptic formation in primary neuronal cultures (H. Zhou et al., 2015). Reciprocally, overexpression of PTEN in primary rat hippocampal neuronal cultures leads to a lack of axonal growth and a decrease in neuronal survival (Jiang, Guo, Liang, & Rao, 2005; C. H. Song et al.,

2010). Interestingly, treatment of primary adult neuronal cultures with rapamycin did not yield a decrease in neurite outgrowth suggesting this growth is independent of mTOR (Christie,

Webber, Martinez, Singh, & Zochodne, 2010).

MicroRNAs which negatively control PTEN have been studied in neuronal cultures and have shown similar effects. In primary mouse neuronal cultures, miR-19a (which was previously

17 known to regulate PTEN in other contexts) downregulates PTEN and axonal outgrowth is subsequently increased (Olive et al., 2009; Zhang et al., 2013). In primary rat cortical neurons, it has been demonstrated that miR-26a reduces PTEN levels and this results in neurite outgrowth promotion (Li and Sun 2013).

Apart from direct regulation of PTEN, there are also indirect effects. Overexpression of miR-182 inhibits PTEN through indirect phosphorylation of the protein (W. M. Wang, Lu, Su,

Lyu, & Poon, 2017). As previously introduced, phosphorylation of PTEN facilitates a closed conformation of PTEN which inhibits activity (Bermudez Brito et al., 2015). These overexpression experiments yield cortical neuronal cultures with an increase in neurite outgrowth (W. M. Wang et al., 2017).

The source of these cell cultures is varied, as is the method of PTEN inhibition but the trend in each that appears evident is that, in mature neurons, neurite outgrowth is increased by inhibiting PTEN. Additionally, as seen in other stem cell cultures, PTEN inhibition yields increases in proliferation and can delay neuronal differentiation (Hill & Wu, 2009; Y. Li et al.,

2017; Z. Shi et al., 2018).

Brain-Specific Mouse Models of PTEN Deficiency

Before discussing the numerous genetically engineered mouse models which are PTEN deficient in the CNS, it is worth noting that there are mouse models for brain tumors that have tumor cells either injected subcutaneously (xenograft) or directly into the brain (orthotopic xenograft). These models are often created in labs with the glioma cells lines that were previously discussed, such as U87MG (Patrizii, Bartucci, Pine, & Sabaawy, 2018). Another method is to utilize tumor cells which are excised from a tumor at the time of surgical resection

(Brabetz et al., 2018; Jensen, Cseh, Aman, Weiss, & Luchman, 2017). One such model is the

18 basis of Chapter 3 of this thesis. These types of models will be briefly discussed in the next section, “Therapeutic Interventions in Brain-Specific Mouse Models.”

Genetically engineered mouse models are an invaluable tool for understanding the initiation, progression, and pathogenesis of diseases. The Cre-loxP system, derived from P1 bacteriophage, for mouse engineering allows for deletions, insertions, and translocations at specific loci in DNA that can be directed at a specific tissue. The enzyme Cre recombinase is used to combine Lox sequences

Here, we review models of PTEN deficiency which specifically target cells of the CNS.

Different promotors, which target different subsets of cells in the CNS, are being utilized. This allows for different CNS-specific models of PTEN deficiency and assessment of specific phenotypes. Additionally, some of the models presented here are inducible which allows for temporal control of PTEN deficiency.

The first model presented here was generated, in part, to understand PTEN deficiency and how it contributes to tumor initiation and growth. It was concluded that PTEN deficiency in the brain is not sufficient to generate brain tumors in the initial model utilized although this has been assessed and confirmed in additional models (Backman et al., 2001; Chow et al., 2011; C.

H. Kwon et al., 2001). It has since been concluded that additional genes must also be aberrantly regulated in concert with Pten in order to generate a brain tumor. Therefore, these complex models will not be thoroughly reviewed here, as dissection of the phenotypes and what factor

(gene) contributes to each component of pathogenesis is difficult to understand. Furthermore, this thesis is focused on PTEN deficiency. Chapter 2 of this thesis highlights a PTEN-deficient model for a brain tumor known as high grade glioma. There are additional perturbations other than Pten which will be discussed in Chapter 2.

PtenloxP/loxP;Glial fibrillary acidic protein (Gfap)-Cre mice were generated to understand the contributions of PTEN to both development and tumorigenesis within the nervous system.

Reduced PTEN is detected in granule cells of the cerebellum, dentate gyrus, and a subset of cortical neurons in these mice (Backman et al., 2001; C.-H. Kwon et al., 2001). This model has pronounced dysplasia of the dentate gyrus (DG) and CA3 of the hippocampus. These mice display both ataxia and seizures beginning at approximately nine weeks (Backman et al., 2001;

C.-H. Kwon et al., 2001). Additional work in this model revealed impairments in learning and memory (Hodges et al., 2018). Interestingly, in a recent study developed to understand the increased risk of bone fractures in individuals with epilepsy, this mouse model also showed decreased bone density as compared to controls but the mechanisms are not well understood

(Lugo, Thompson, Huber, Smith, & Kwon, 2017). This model will be a partial focus in the thesis work of Chapter 4.

Additional models have been developed which utilize alternative Gfap-Cre transgene mouse models for specific Pten deletion in the nervous system (M. Su et al., 2004). To elaborate, GFAP is an intermediate filament which is predominately found in astrocytes (Jacque et al., 1978). This makes the model ideal for astrocytic cell targeting; however, certain transgene sequences are known to direct activity in neurons (M. Su et al., 2004). Gfap regulatory elements have been used to develop promotors which target expression in different subsets of both neurons and astrocytes (Y. Lee, Messing, Su, & Brenner, 2008). Thus, it is important to assess

Cre expression in each Gfap-Cre mouse model so the context of Pten deletion and the cells affected are understood.

An independently generated PtenloxP/loxP;Gfap-Cre mouse model utilized by Fraser et al showed PTEN deficiency in many pyramidal neurons and granule cells of the dentate gyrus

(Fraser et al., 2004). Brain hypertrophy associated with both increases in cell migration and cellular size was attributed to a decrease in survivability in this model (Fraser et al., 2004).

PtenloxP/loxP;h (human)Gfap-Cre mice show high levels of PTEN deficiency in the

Bergmann glial cells and nearly complete deletion occurs in the granule neurons of the cerebellum (Yue et al., 2005). Due to severe macrocephaly, these mice are only viable for approximately 21 days (Yue et al., 2005).

PtenloxP/loxP;Gfap-CreER-A mice, an inducible model, were used to investigate the effect of Pten loss in mature astrocytes and assess if Pten was sufficient to generate cancer within the brain (Chow et al., 2011). Interestingly, Pten deletion induced in developmentally mature mice did not disrupt brain structure as seen in similar models, nor did it initiate tumor growth. The addition of a second tumor suppressor (p53 or Rb1) was sufficient to generate high grade gliomas (Chow et al., 2011). An extension of this mouse model with additional deleted genes will be described in Chapter 2 of this work.

Nestin promoter-driven Cre mice were crossed with PtenloxP/loxP mice to investigate PTEN function in early brain development (Groszer et al., 2001). This model reported PTEN deficits by mid-gestation and embryonic brain size is increased as compared to litter mate controls by embryonic day (E)14. These mice die shortly after birth and histological examination showed an increase in both cell size and number (Groszer et al., 2001).

PtenloxP/loxP; engrailed 2(En2)-Cre mice have Pten deletion in the developing cerebellum and mice exhibited an increased cerebellum size by E15.5 (Marino et al., 2002; Zinyk, Mercer,

Harris, Anderson, & Joyner, 1998). Mice exhibited ataxia, lethargy, and a reduction in balance.

In an extension of this research, Marino et al. generated an additional mouse model that utilized

L7cre756, which selectively deletes Pten in Purkinje cells. This model displayed only minor enlargements of Purkinje cells and no clinical manifestations (Marino et al., 2002).

Kwon et al generated an additional PTEN-deficient mouse model by utilizing neuron- specific enolase (NSE)-Cre in order to selectively delete Pten in a subset of differentiated

21 neurons, predominately in layers III, IV, and V in the cerebral cortex and the CA3, dentate gyrus granular layer (GL), and the polymorphic layer (PML) of the hippocampus (Kwon et al., 2006).

This model showed not only the histological abnormalities previously noted in similar PTEN- deficient models, but also multiple behavioral abnormalities including decreased social interactions. This work further strengthened the reported clinical findings which correlate PTEN mutations to a subset of individuals with autism (Butler et al., 2005).

PtenloxP/loxP; alpha-calcium-calmodulin kinase II (CamKIIα)-Cre mice have a subset of excitatory neurons that are PTEN-deficient within the CA1 and CA3 of the hippocampus

(Sperow et al., 2012). Attributed to the late loss of PTEN (postnatal (P)14)), these mice are reported to have a normal brain morphology, but a recent paper showed an increase in brain weight (C. J. Chen et al., 2019; Sperow et al., 2012). This model displayed an impairment in both synaptic plasticity and spatial memory. These mice developed seizures and died at approximately 11 weeks of age (McMahon et al., 2012). This model will be utilized extensively in

Chapter 4 of this thesis.

PtenloxP/loxP; Glioma-Associated Oncogene Homolog 1 (Zinc Finger Protein) Gli1-Cre estrogen receptor tamoxifen 2 (ERT2) mice were generated in order to assess the contribution of newly born dentate granule cells to temporal lobe epilepsy (LaSarge, Pun, Muntifering, &

Danzer, 2016; Pun et al., 2012). In this inducible model, Cre expression occurred in neuronal progenitor cells so that only newly born cells were targeted for Pten deletion (Pun et al., 2012).

Hippocampal slices prepared from this model exhibit multiple circuit abnormalities, including increased field excitatory post-synaptic potential and multiple population spikes following perforant path stimulation. Mice developed spontaneous seizures (Pun et al., 2012).

This model displayed a multitude of phenotypes dependent upon the spatial and temporal control of Pten deletion. With varied time and/or dose of tamoxifen, the granule cells

22 affected could be altered, changing the percentage of PTEN-deficient cells. With Pten deletion rates ranging from less than 1% to greater than 30% in hippocampal dentate granule cells, phenotypes varied and severity of phenotypes related to quantity of cells that were PTEN- deficient (Arafa, LaSarge, Pun, Khademi, & Danzer, 2019; LaSarge, Pun, Gu, Santos, &

Danzer, 2019).

The PtenloxP/loxP;2',3'-Cyclic Nucleotide 3' Phosphodiesterase (Cnp1)-Cre mouse model conditionally inactivated Pten in oligodendrocytes and Schwann cells (Goebbels et al., 2010).

Hypermyelination was evident in adult mutant mice and most white matter tracts were reported as enlarged when compared to litter mate controls (Goebbels et al., 2010).

PtenloxP/loxP; Oligodendrocyte transcription factor 2 (Olig2)-Cre mice were developed in order to investigate Pten loss in oligodendroglial lineage and subpopulations of neural progenitor cells (NPCs) (Maire et al., 2014). Common phenotypes of this model included hypermyelination and brain enlargement due to increased cellular proliferation and changes in the cellular differentiation, as cells remained less differentiated. At approximately six months, these mice had progressive ataxia, decreased motor function, bilateral hind limb paralysis, and premature death (Maire et al., 2014).

The described work above in the diverse mouse models partially explains how factors, such as spatial and temporal expression, can play an important role in clinical manifestations; however, addressing how environmental factors and additional mutations contribute remains elusive. Of note, the mouse models described here mimic somatic mutations. Whereas, human

PTEN mutations associated with neurological disorders are often germline (Nelen et al., 1997).

This difference may have contributed to the pronounced seizure phenotype which was seen in mouse models, but often absent in humans (Biesecker et al., 1999; Butler et al., 2005; Eng,

2000; Marsh et al., 1999; Nelen et al., 1999; Pilarski & Eng, 2004; Varga, Pastore, Prior,

Herman, & McBride, 2009). Page et al created a Pten haploinsufficient mouse model to better

23 mimic germline mutations and the phenotypes associated with germline mutation disease

(Page, Kuti, Prestia, & Sur, 2009). Both male and female mice had brain overgrowth, but only females were reported to have deficiency in social behavior. In order to assess if this was accurate, additional social behaviors beyond social approach behavior would need to be assessed. There was no report of a seizure phenotype in this model, further supporting the potential link between somatic mutations and seizures (Page et al., 2009). Additional haploinsufficient models confirmed a progressive macrocephaly phenotype, increased repetitive behaviors, and no seizure phenotype (Y. J. Chen, Huang, Sejourne, Clipperton-Allen, & Page,

2015; Clipperton-Allen & Page, 2015).

Therapeutic Interventions in Brain-Specific Mouse Models

Therapeutic Approaches in Mouse Models of Brain Tumors

Orthotopic models for PTEN-deficient brain tumors have shown that inhibition of PI3K significantly reduces downstream signaling as assessed via pAkt (Salphati et al., 2016).

Downstream effectors of the PI3K pathway, pS6 and p4EBP1, were also reduced, but not to the striking levels seen with pAkt (Figure 3) (Salphati et al., 2016).

Orthotopic models of both established cell line and patient-derived brain cancer cells lines showed a significantly increased survival time with the use of mTOR inhibition (Fan,

Nicolaides, & Weiss, 2018). The driving mutations were not stated and it was therefore not known if or what kind of mutations in the PI3K pathway were present (Fan et al., 2018).

A survival study utilizing two PTEN-deficient orthotopic models has shown that a dual

PI3K and mTOR inhibitor increased survival, but not significantly. Interestingly, when this compound was given in combination with temozolomide (an alkylating agent and current standard of care in gliomas) survival was significantly increased (Koul et al., 2017).

Therapeutic Approaches in Mouse Models of Neurological Disorders

PTEN-deficient mouse models have not only provided insight into the importance of mutation context, they represent model systems that can be used to assess therapeutic modalities. Given the known dysregulation to PI3K signaling in conjunction with PTEN deficiency, a frequently exploited strategy has been to administer small molecule inhibitors in order to reduce PI3K and downstream PI3K signaling. In most cases investigators used inhibitors of mTOR, such as rapamycin or everolimus, as these were already approved for use in humans for other indications (Ljungberg, Sunnen, Lugo, Anderson, & D'Arcangelo, 2009;

Sunnen et al., 2011; J. Zhou et al., 2009).

Ljungberg et al subjected the previously described PtenloxP/loxP;Gfap-Cre model to short- term inhibition of mTOR with rapamycin (Ljungberg et al., 2009). Individual PTEN-deficient neurons are known to be hypertrophic and the administration of rapamycin significantly reduced the size of the neuronal body in six-week old PtenloxP/loxP; Gfap-Cre mice. Furthermore, rapamycin significantly reduced the time spent in epileptic activity in this model (Ljungberg et al.,

2009).

Treatment with rapamycin in PtenloxP/loxP;Gfap-Cre mice, given on different therapy regimens, blocks aberrant mossy fiber sprouting (Sunnen et al., 2011). Rapamycin also reduced seizures and increased survivability during and directly after treatment, and as expected, the longer therapy regimen yielded the most favorable increase in survivability (Sunnen et al.,

2011).

Zhou et al utilized rapamycin in the PtenloxP/loxP; NSE-Cre model (J. Zhou et al., 2009).

Using long-term therapy, a multitude of macrocephaly phenotypes, including soma enlargement, mossy fiber tract thickness, and dentate gyrus enlargement were rescued; however, rapamycin

25 administration did not prevent or correct excessive axonal projections. Anxiety, seizure time, and seizure duration were also reduced (J. Zhou et al., 2009).

To our knowledge, so far, there is only one study using a pan-PI3K inhibitor, which targets the most direct counteractor of PTEN’s lipid phosphatase activity. In this study, NVP-

BKM120 (BKM120), a pan-PI3K inhibitor, was utilized to assess the effects of PI3K inhibition in the context of brain-specific PTEN deficiency in PtenloxP/loxP;Olig2-Cre mice (Maire et al., 2014).

After one month of BKM120 administration that began at P21, Akt phosphorylation was reduced in the subventricular zone (SVZ) and resulted in no significant changes in brain structure, myelination or neurogenesis (Maire et al., 2014).

Rapamycin and PI3K inhibition have rescued a multitude of phenotypes associated with

PTEN-deficient mouse models, but the extent of rescue has shown variability and is likely dependent on the drugs (combination of drugs and concentration), the length of therapy administration, as well as the initiation of therapeutic intervention. Using retroviral injections directly targeting the hippocampal dentate gyrus of neonatal mice, Getz et al assessed precise temporal changes in neuronal migration and hypertrophy in the context of PTEN deficiency

(Getz, DeSpenza, Li, & Luikart, 2016). They administered rapamycin at different time points to understand if these phenotypes are solely preventable or also reversible. Interestingly, this study showed that rapamycin could both prevent and reverse neuronal hypertrophy, but could only prevent abnormalities in migration (Getz et al., 2016).

MTOR is known to play an important role in two protein complexes which are activated differently and affect different downstream targets, mTORC1 and mTORC2. Rapamycin is considered an inhibitor of the mTORC1 complex, although there is research which suggests long-term administration may also inhibit mTORC2 (Sarbassov et al., 2006). In PtenloxP/loxP;

CamKIIα-Cre mice, genetic deletion of the mTORC2 complex but not the mTORC1 complex significantly reduced seizures, increased survivability, and improved both memory and social-

26 like behaviors (C. J. Chen et al., 2019). Inhibition of mTORC1 reduced only brain weight and none of the additional phenotypes assessed, such as the seizure phenotype (C. J. Chen et al.,

2019). Macrocephaly is often associated with increased risk of seizures, so it is interesting that varied mTORC complexes appeared to be responsible for each phenotype (Baple et al., 2014).

A small molecule inhibitor which targets mTORC2 is not commercially available so Chen et al developed and utilized short, synthetic, single-stranded antisense oligonucleotides (ASO) which targeted rictor (a protein found in the mTORC2 complex). A single intracerebroventricular (ICV) injection improved both the epilepsy phenotype and behavioral abnormalities (C. J. Chen et al.,

2019). These results are interesting and additional studies will be needed in order to further elucidate some of these findings. For instance, is the crosstalk between the mTORC1 and mTORC2 complexes responsible for the positive phenotypes reported in this work (J. Xie &

Proud, 2014)? Additionally, would mTORC2 inhibition benefit neurological disorders and brain tumors in which the PI3K pathway is increased? These aspects of the mTORC complexes and inhibition needs to be more thoroughly clarified.

In this thesis, another therapeutic strategy which has been successfully applied to

PTEN-deficient cancer models will be assessed in a mouse model of neuron-specific PTEN deficiency. It is known that, in cancers which contain a driving mutation in PTEN, inhibition of one of the three class 1A catalytic isoforms, p110β, yields a decrease in tumor growth or stops tumor initiation (Jia et al., 2008; Schmit et al., 2014; Wee et al., 2008; Yuzugullu et al., 2015).

We will assess if p110β inhibition rescues neuronal phenotypes and the results of this work will be thoroughly discussed in Chapter 4 (White, Tiwari, MacLeod, Danzer, & Gross, 2020).

Therapeutic Interventions in Clinical Studies

Clinical Studies in Cancer

The clinical data for cancer studies which utilize inhibition of the PI3K pathway is difficult to interpret. Most initial studies did not stratify patients that are receiving the novel therapy by the genetic composition of the tumor. Therefore, many patients may not have had driving mutations in the PI3K pathway and in the clinical trial summary data available for many studies

(clinicaltrials.gov), driving mutations may not be discussed. Table 3 summarizes completed clinical trial data, as well as reported tumor responses. There are several ongoing cancer studies which stratify patients with mutations in the PI3K pathway, including PTEN mutations, but some of these studies are utilizing therapy which is not targeting the PI3K pathway.

Additionally, most ongoing studies have not reported preliminary results. All reported data for oncology clinical trials were obtained from ClinicalTrials.gov.

Clinical Studies in Neurological Disorders

In conjunction with multiple studies reporting the partial or full rescue of phenotypes associated with PTEN-deficient mouse models, clinical investigation for neurological disorders is ongoing for the use of mTOR inhibition in individuals with aberrant regulation of PI3K signaling.

Cowden syndrome is one of the PHTSs and is classified by an inactivating germline mutation in PTEN (Hobert & Eng, 2009). Sirolimus, an inhibitor of mTOR, was given to 18 individuals with Cowden syndrome and there was a reported improvement in cerebellar function score, which assesses muscular coordination, after one month. Characteristic skin and gastrointestinal lesions were also reported as improved (Komiya et al., 2019).

There is an ongoing clinical trial, expected to publish results within approximately six months, which is utilizing the mTOR inhibitor, RAD001 (everolimus) in individuals with PHTS.

Patients receive(d) either RAD001 or placebo for six months, followed by six months of open-

28 label mTOR inhibitor use. The main assessment for this study is neurocognition and behavior

(ClinicalTrials.gov Identifier: NCT02991807). There are a multitude of ongoing clinical trials which are assessing mTOR inhibition in individuals with PTEN deficiency which are less progressed.

Tuberous sclerosis complex (TSC) is a disorder in which either of the TSC proteins

(TSC1 or TSC2) is dysregulated. Similarly, as seen with PTEN deficiency, TSC dysregulation ultimately leads to an increase in mTOR. This makes mTOR inhibition a strong candidate for therapy. Administration of the mTOR inhibitor everolimus in patients with TSC-related epilepsy yielded more than 50% reduction in seizure frequency in 12 of 20 patients. Five of 20 patients experienced a 25-50% reduction (Krueger, Wilfong et al. 2013). The differences between individuals who favorably respond and those that do not respond is not well understood, but should be analyzed in order to develop the most efficient treatment strategies.

Concluding Remarks

PTEN is a complex protein which contributes to many growth disorders, including cancer and several disorders in the brain. Here, in addition to description of its molecular characteristics and functions, we have addressed the historical and structural aspects of this protein and have provided a background on the discovery of PTEN to illustrate its importance in the context of both health and disease. Additionally, we have discussed the structural components as well as the multitude of ways in which PTEN may be regulated. Mutational regulation of PTEN is only a minor component of PTEN deficiency. Therefore, it is likely that PTEN deficiency contributes to an even larger population of diseases than what is currently understood.

It has been established that PTEN regulates the ratio of PIP2 to PIP3. Here we also discuss alternative functions of PTEN. What is yet to be elucidated is if there are feedback loops

29 that connect the alternative functions of PTEN to the most acknowledged and described function of PTEN as a negative regulator of PI3K signaling.

As described, there are a multitude of PTEN-deficient mouse models which are specific to the CNS. These models have demonstrated that both the cell type, as well as the stage in development in which they are affected can severely alter phenotypes and severity of phenotypes. A similar phenomenon is noted in many brain tumors, meaning the combination of additional mutations found in conjunction with PTEN mutations can alter the characteristics of the tumor (Chow et al., 2011; Knobbe, Merlo, & Reifenberger, 2002).

Work is ongoing in cellular models, mouse models, and humans which has often targeted mTOR inhibition. In mice, this work has often decreased or ceased tumor growth and autism-associated phenotypes. To date, mTOR inhibition has initially shown some improvement in clinical assessment, but there are general concerns about long-term usage of mTOR inhibition in patients, specifically children. MTOR is a ubiquitously expressed protein which plays many important roles within the body and indiscriminate inhibition could potentially lead to side effects throughout the body (Baselga et al., 2012; Bellmunt, Szczylik, Feingold, Strahs, &

Berkenblit, 2008; Willemsen et al., 2016). Therefore, alternative strategies, especially in neurological disorders that require chronic treatment, need to be explored. Additionally, given the many feedback loops which are connected to the PI3K pathway, mTOR may become reactivated (Efeyan & Sabatini, 2010). For this reason, in multiple disorders, including both brain tumors and autism, combination approaches are being assessed (Conciatori et al., 2018).

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Figures and tables

Figure 1-The Domains of PTEN. The PTEN protein domains: the N-terminal domain, the phosphatase domain, the C2 domain, the C-terminal tail, and the PDZ binding motif.

Figure 2-Mutations of the PTEN Gene. A schematic of the PTEN gene and its five components shows the distribution of mutations. PHTS mutations are represented in pink

(A39P, N48K, L108P, L112P, R130L). Autism spectrum disorder mutations are in gray (D22E,

H93R, H123Q, R173H, Y178X, F241S, D252G, W247L, N276S, D326N, H118P, E157G, L23F,

Y65C, Y68H, I101T, I122S and L220V). Cancer mutations are brown (D24G, D92A, R130G,

M134R, M205V, L345V).

Figure 3- PTEN is the main negative regulator in the PI3K pathway. There are 4 class 1 PI3K catalytic subunits (p110-purple): p110α, β, δ, and γ. Each isoform has both redundant as well as distinct functions in the brain. The lipid phosphatase activity of PTEN (blue) negatively regulates class I PI3K downstream signaling by dephosphorylating PIP3.

Proteins Genes Description AKT-1, Ser/Thr protein akt-1* Pleckstrin Homology. Domain kinase for PIP3 Muscle M-line assembly unc-89 Pleckstrin Homology. protein Domain, PIP2/3 14-3-3 proteins par-5**, ftt-2** Likely PHDs binding PIP2 or PIP3 Disorganized muscle protein dim-1 Associates with a PHD 1 protein Vitellogenins 2, 3, 5, 6 vit-2*,-3*,-5*,-6* LDLs related to ApoB100

HSP chaperones: HSP70C, hsp-3+, -4+, -60+; daf-21+ Chaperonins needed for HSP70D, HSP60; misfolded proteins; HSP-60 is DAF21/HSP90 mitochondrial HSP70A hsp-1 V-type proton ATPase s.u.’s+ vha-12+, -13+, -15 Vacuole H+-translocating ATPase Fatty-acid binding proteins far-1, −2; lbp-6* Lipid transporters; lbp-6 KD is LL Tubulin alpha-2, beta-2 tba-2+, tbb-2+ Structural protein chains Protein disulfide isomerase 2 pdi-2+ Role in ER folding oxidized proteins rRNA 2′-O-methyltransferase fib-1 Fibrillarin, part of U3 SnoRNP Lamin-1 lmn-1** Nuclear envelope structural protein T-complex protein 1 s.u. ϵ cct-5+ Chaperonin complex Alpha Enolase enol-1+,*,** RNAi alters LS Adenosylhomocysteinase ahcy-1+ Interacts w. CCT, UBA-1, UBQ-2 Pyruvate carboxylase 1 pyc-1** Regulating enzyme for glucose & lipid metabolism. Methylcrotonoyl-CoA F02A9.4 Function predicted, not carbox.β proven 60S ribosomal proteins rpl-4, 5, 7–10, 20, 36 Other 60S proteins not PIP3- spec. 40S ribosomal proteins rps-7, 12, 13, 19,23,25 Other 40S proteins not PIP3- spec. Cullin-associated, NEDD8- cand-1* Assembles SCF (SKP1- dissociated protein 1 CUL1-F-box) /E3-ubiquitin ligase complexes Rad-50 rad-50 HR-directed DNA DS-break repair Translationally-controlled tct-1 ER protein needed in tumor protein homolog development, growth, locomotion, reproduction

Dynein heavy chain, dhc-1 Places microtubule cytoplasm organizing control Peroxiredoxin prdx-3 Oxidative-stress response Acetyl-coA acetyltransferase, kat-1 Fatty-acid β-oxidation, via mit. Sir2 Proteasome α subunits pas-1,-3,-5,-6,-7 Proteasome structural/regul. SU's

Table 1- Proteins that bind to PIP3 through a PH domain interaction. Each protein has been verified though a minimum of two experiments. Akt is noteworthy as it is upstream of mTOR activation. The other proteins listed are noteworthy because they are theoretically affected by PTEN deficiency yet not well described as compared to Akt.

Channel Name Channel Classification PIP2 Regulation KCNQ Voltage gated K+ Activation KV Voltage gated K+ Removal of inactivation HCN Voltage gated K+ Activation HERG Voltage gated K+ Inactivation CNG Voltage gated K+ Inhibition CAV2.1 Voltage gated Ca+ Activation CAV2.2 Voltage gated Ca+ Activation ENaC Renal epithelial Na+ Activation IP3 receptor Ca+ release Inhibition Ryanodine receptor Ca+ release Activation Kir1.1 Inward-rectifier K+ Activation Kir2.x Inward-rectifier K+ Activation Kir3.x Inward-rectifier K+ Activation Kir5.1 Inward-rectifier K+ Activation Kir7.1 Inward-rectifier K+ Activation Kir6.x Inward-rectifier K+ Activation TASK1 Two-P domain K+ Activation TASK3 Two-P domain K+ Activation TREK1 Two-P domain K+ Activation TRAAK Two-P domain K+ Activation TRPL TRP Channel Activation TRPV1 TRP Channel Context-dependent activation or inhibition TRPV5 TRP Channel Activation TRPM4/8 TRP Channel Activation TRPM5/7 TRP Channel Activation TRPC1/5/6/7 TRP Channel Activation NCX Transporter Activation NHE1-4 Transporter Activation NME Transporter Activation PMCA Transporter Activation

Table 2- Ion Channels and Transporters Regulated by PIP2. PIP2 often activates but may, in some contexts, inhibit ion channels.

Drug Combination Phase Patient #/Response BEZ235 1/2b 15:40% SD BKM120 1a/MTD 66: 42%SD, 5%PR, 30%DR BKM120 1 10: 80% BKM120 Trametinib 1b 113: 6.2%PR to CR PX-866 1 60: 22% CR PX-866 1 31: 22% CR PX-866 Docetaxel 1 35: 63% SD, 6% PR PX-866 2 33: 24% SD, 3% PR XL147 1/MTD 75: 17% SD XL147 Erlotinib 1 27: 51.9% SD, 3.7% PR XL147 1 69: 43.9% SD, 11% PR

Table 3-Cancer clinical trials which utilize inhibition in the PI3K pathway. BEZ235 is a

PI3K and mTOR inhibitor. BKM120 is a pan PI3K inhibitor. PX 866 is a pan PI3K inhibitor with moderate inhibition of mTOR. XL147 is a pan PI3K (only mild inhibition of p110β). Varied responses are reported. SD=stable disease. PR=partial response.

Dissertation objectives

The overall goal of this dissertation work was to assess some of the challenges in the fields of PTEN-deficient cancer and autism. My overall hypothesis is that inhibition of PI3K will decrease aberrant downstream PI3K signaling and decrease aberrant phenotypes in mouse models for both high grade gliomas, autism, and epilepsy. My aims were as follows:

Aim 1: Assess downstream PI3K signaling in QKO tumors exposed to PI3K inhibition.

Subsequently assess survival properties both through molecular assessment as well as through survival study analysis. Assess molecular variance within tumors which have been treated using

PI3K pathway inhibition.

Aim 2: Create a pediatric high grade glioma model and assess PI3K pathway inhibition in the tumors generated in the model.

Aim 3: Assess molecular signaling effects, seizures, and macrocephaly of p110β inhibition in the context of neuron-specific PTEN deficiency.

Chapter 2: PI3K inhibition yields varied tumor response in a novel mouse model for high

grade glioma

Angela White, Ralph Salloum, Annmarie Ramkissoon, Amanda Stang, and Lionel L.M. Chow

My primary contribution was data generation and analysis in Figures 3 (E-F), 4, 5, and 6

Abstract

High grade glioma (HGG) has a dismal prognosis and patient outcome has improved only marginally in the last 20 years despite aggressive research and improvements in therapy and supportive care. One hurdle in the field is the lack of molecularly accurate animal modeling systems which can be utilized to both understand the disease and test novel therapeutics. Here, we present a novel mouse model, Gfap-CreER; PtenloxP/loxP; Tp53loxP/loxP; Rb1loxP/loxP; p107-/-

(henceforth QKO), and characterize response to treatment with Temozolomide and radiation therapy, which are standard therapies used in HGG. Our assessment shows the tumors respond in a similar manner as human tumors. Next, we subjected the model to inhibition of the

Phosphoinositide 3-kinase (PI3K) pathway, given its conditional deletion of the negative regulator of the PI3K pathway, Pten. Interestingly, we found a subset of tumors which respond favorably to this therapeutic approach and a majority which do not. Preliminary assessment of these tumors suggest tumors with a greater population of progenitor cells do not react as favorably as tumors with a more differentiated phenotype.

Introduction

High grade glioma (HGG) is the most prevalent form of primary, malignant brain tumor.

Median survival for glioblastoma, the most aggressive form of the disease, is only 14.6 months and long term survival is 9.8% for patients who undergo radiation and Temozolomide (the current treatment standard) (Buckner, 2003; Johnson & O'Neill, 2012; Stupp et al., 2002; Stupp et al.,

2005; Wen & Kesari, 2008). Although research has greatly improved our knowledge of HGG biology, treatment options have only modestly progressed over the past several decades.

Developing in vivo models which accurately recapitulate molecular biology of HGG is necessary to develop and validate improved therapy options.

Analysis of patient-derived tumor samples has determined that HGG can be divided into five distinct gene expression subgroups: proneural (G-CIMP and non G-CIMP), neural, classical, and mesenchymal (Noushmehr et al., 2010; Phillips et al., 2006; Verhaak et al., 2010). Gene expression analysis, microarray analysis to identify copy number differences, and analysis to identify common mutations has collectively concluded that there are three core pathways dysregulated in gliomagenesis: Rb-mediated control of the cell cycle, p53 signaling, and the

RTK/PI3K/AKT pathway (Cancer Genome Atlas Research, 2008; Parsons et al., 2008). These pathways are dysregulated in each of the gene expression subtypes.

The genes and pathways dysregulated in gliomagenesis have been described in both the brain and more specifically HGG. Rb1-mediated cell cycle defects result in an increase in proliferation in the embryonic brain (Ferguson et al., 2002). Similar defects are known to play a role in many types of cancer, including HGG. The tumor suppressor TP53 is known to be necessary for the induction of apoptosis in neural progenitor cells (Hede, Nazarenko, Nister, &

Lindstrom, 2011; Jacobs, Kaplan, & Miller, 2006). TP53 deficient neuronal cells are more resistant to irradiation and chemotherapy (Tedeschi & Di Giovanni, 2009). The RTK/PI3K/AKT pathway is activated in many cancers, including HGG (Cancer Genome Atlas Research, 2008; Fan & Weiss,

2011). This pathway is known to control many cellular functions central to tumor development, such as proliferation and metabolism (Bader, Kang, Zhao, & Vogt, 2005; Engelman, Luo, &

Cantley, 2006; Vivanco & Sawyers, 2002).

Proneural HGG is a well-represented subtype in genetically engineered mouse models

(GEMMs) (Halliday et al., 2014; Lei et al., 2011; Saito et al., 2014; Sonabend et al., 2013). Median survival is longer than other subtypes, such as mesenchymal (Cooper et al., 2010; Phillips et al.,

2006). Mesenchymal glioblastoma displays genetic lesions of chromosomes 7 and 10. These aberrations are not generally present in the proneural subtype (Phillips et al., 2006). Mutations of the IDH1 gene are present in approximately 30% of the proneural gene expression subtype and generally not mutated in mesenchymal tumors (Noushmehr et al., 2010; Verhaak et al.,

2010). PDGFRA is amplified almost exclusively in the proneural HGGs (Noushmehr et al., 2010).

NF1 is mutated in 37% of mesenchymal glioblastomas and 5% of proneural glioblastomas

(Verhaak et al., 2010). These genetic differences potentially effect the pathways which drive disease pathology and illustrate why models for each subtype are needed to study tumorigenesis and subsequently formulate therapies for patients.

Most in vivo HGG research has been conducted in xenograft models. Although insight has been gained from these models, there are aspects of tumor biology that xenografts are not able to recapitulate. These include tumor initiation and progression, stromal interactions, and the roles of the immune system in tumorigenesis and treatment (Chen, McKay, & Parada, 2012;

Fomchenko & Holland, 2006). GEMMs offer an opportunity to study all these aspects of tumorigenesis and may offer a more accurate approach for preclinical studies.

GEMMs for medulloblastoma, a fast-growing high grade tumor of the cerebellum, have been utilized to test novel therapeutics. Romer, et al pioneered this work by describing how suppression of the Shh pathway affected tumor growth in Ptc1+/- p53-/- medulloblastoma (Romer et al., 2004). This and similar work has led to several clinical trials, advancing patient care

(Catenacci et al., 2015; Gajjar et al., 2013; Robinson et al., 2015). Comparable approaches for

HGG have been more limited. Pitter, et al generated a PDGF-B driven GEMM and tested novel therapeutics which target AKT and mTOR (Pitter et al., 2011). They subsequently generated additional PDGF-driven glioma models and demonstrated that temozolomide could be supplemented with the novel AKT inhibitor perifosine (Momota, Nerio, & Holland, 2005). Such approaches to preclinical testing may offer a more predictive model system in which to study HGG and is like the approach we will present.

Here, we will introduce a novel mouse model for HGG and utilize a similar approach that was described for medulloblastoma to test the effects of PI3K inhibition on HGG.

Materials and Methods

Mice

Gfap-CreER; PtenloxP/loxP; Tp53loxP/loxP; Rb1loxP/loxP; p107-/- mice were generated as previously described in Chow, et al. (2011).

Mouse Tissue Collection

Mice were anesthetized with 2,2,2-Tribromoethanol (Sigma-Aldrich) and perfused with 1x phosphate buffered saline (PBS) for tissue exsanguination. Brains were removed and examined for tumors. If tumors were grossly identified, a portion of the tumor was removed and immediately frozen on dry ice, along with a normal cortex sample. The remaining brain (or total brain if no tumor was grossly identified) was immersed in 4% paraformaldehyde (4% PFA) in 1x PBS for 24 hours. Frozen tissue was stored at -80, for protein or nucleic acid preparations. PFA preserved tissue was processed and embedded in paraffin. 5 um sections were cut for histology and immunohistochemistry IHC.

PI3K Inhibitor(s) Formulation

PX-866 (Oncothyreon) was prepared in 50 mM hydroxypropyl-cyclodextrin at a concentration of 0.3 mg/ml. Aliquots were prepared weekly, stored at -80, and thawed daily prior to administration. Rapamycin (LC-Laboratories) stock was prepared in DMSO at a concentration of 50mg/ml. Aliquots were prepared weekly and stored at -20. Aliquots were thawed daily and diluted with 5.2% Tween 80 in ddH2O to a concentration of 2.5mg/ml prior to administration.

Histology and Immunohistochemistry

Sections were deparaffinized with xylene, passed through graded alcohols, and stained with hematoxylin and eosin (H&E) reagents. IHC sections were similarly deparaffinized and passed through graded alcohols to water. Antigen retrieval was achieved by immersing slides in

72 citrate buffer and boiling slides. Next, slides are immersed in 1% H2O2 for 30 minutes and subsequently blocked for minimum of an hour in 10% goat serum. Supplemental table 1 lists the antibodies used for IHC.

Western Blot

Tumor or normal tissue were lysed using 2D lysis buffer (BioRad) with Mini-Protease

Inhibitor Cocktail (Roche) and PhosSTOP Phosphatase Inhibitors (Roche) added. Proteins were separated by electrophoresis on NuPAGE 4-12% Bis-Tris Gels (Invitrogen). Gels were transferred to blotting paper (Invitrogen). Blots were blocked with 5% milk for one hour and the primary antibody added overnight. Detection was achieved using either Super Signal (Fisher) or ECL

(Fisher).

Statistical Analysis

Specific signals on western blots were quantified densitometrically using NIH ImageJ software (Bethesda, Maryland, USA). Signal intensities of phospho-AKT, phospho-S6K, PTEN, and Puromycin were normalized to AKT or β-Tubulin signal on the same blot. Phospho-S6 signal intensities were normalized to the signal intensities of total PDK1 and S6 on the same blot, respectively. The average of the duplicates was counted as one data point.

For analysis of biomarkers, tumors were quantified (500 cells/tumor) and entered into GraphPad

Prism 7.01. Correlation and linear regression analysis were both performed.

For all tumor sphere assays, analysis was generated by Graphpad Prism 7.01. All experiments were carried out in triplicate or greater. All p values are two-tailed and p values less than 0.05% were considered significant. Data are shown as mean +/- SD.

Results

QKO tumors closely mimic human HGGs both genetically and histologically.

We generated Gfap-CreER; PtenloxP/loxP; Tp53loxP/loxP; Rb1loxP/loxP; p107-/- (henceforth QKO) mice. Cre was induced in mature mice at 5-6 weeks of age with tamoxifen (9mg/40g daily for 3 days). Mice were monitored and sacrificed at moribundity. We detect multiple tumors, which originate in various locations of the brain with H&E staining (Figure 1A,B). QKO tumors display heterogeneity, a common characteristic of human HGG. Anaplastic astrocytoma is represented in the model (Figure 1C). Pseudopallisading necrosis, a common characteristic of glioblastoma multiforme (GBM) which is associated with poor prognosis, is present in a subset of QKO tumors

(Figure 1D). Areas of leptomeningeal disease (Figure 1E) and perivascular spread (Figure 1F) are common characteristics of human tumors seen in the QKO model.

QKO tumors were classified into either the mesenchymal, proneural, or proliferative gene expression subtype as defined by gene expression data (Figure 1G). Global copy number aberrations were assessed via Microassay-based Comparative Genomic Hybridization (aCGH)

(Figure 1H). Losses in chromosomes 7 and 8 (boxed) are frequent in the QKO model. The minimal region of loss on mouse chromosome 7 is syntenic to human chromosome 19q where loss of heterozygosity has been noted to occur in human HGG.(Bigner et al., 1988)

We examined the brains of QKO mice prior to moribundity to determine when tumors begin to develop. Six weeks after tamoxifen injections were administered, 100% of mice have tumors present (Supplemental Figure1). At approximately eight weeks, mice began to display visible signs of illness (dehydration, body weight loss, etc.) and will quickly begin to succumb to disease shortly after this time (Figure 1I). The addition of the p107-/- allele reduces the time to moribundity as compared to PtenloxP/loxP; Tp53loxP/loxP; Rb1loxP/loxP model, as assessed by Kaplan-Meier survival

(Figure 1I).

The immunophenotype of QKO tumors was assessed by immunohistochemistry (IHC).

Proliferation was assessed by KI67 IHC. Tumors display a high proliferative index (Figure 1J).

PAKT and pS6 staining is increased in tumors, indicating an increase in PI3K signaling (Figure

1K, L). Glial fibrillary acidic protein (GFAP) which is indicative of intermediate filament protein in astrocytes is found in all QKO tumors (Figure 1M). PTEN staining is high in normal brain tissue but low in QKO tumors, as is expected with the PtenloxP/loxP allele (Figure 1N).

These data suggest that the QKO model closely mimics human HGG histologically and genetically. Importantly, this model generates tumors with 100 percent penetrance in a short, predictable time frame, making it optimal for preclinical evaluation.

Radiation and temozolomide response in QKO tumors

Next, we wanted to understand if QKO tumors display a similar response when subjected to a treatment regimen comparable to that given to patients with HGG. Mice were injected with tamoxifen and monitored for eight weeks. Mice were then exposed to radiation daily on a schedule of 5 days on, 2 days off for 2 weeks (5/2 x2) (Figure 2A). These mice also received either temozolomide or vehicle. Mice were next sacrificed for sample collection. Histological examination of tumors showed changes in morphology after radiation treatment, as seen in human tumors (Figure 2B). KI67 decreased after radiation exposure, with or without temozolomide (Figure 2C). Quantification of these IHCs determined that radiation (with and without temozolomide) yields a significant decrease in KI67 positive cells (Figure 2E). Cleaved caspase-3 did not significantly change after radiation exposure. (Figure 2D,F).

Next, we assessed the potential survival benefits of temozolomide and radiation. Mice were treated as described above and monitored daily until symptoms developed requiring them to be sacrificed. The combination of temozolomide and radiation produces a modest increase in

75 survival as compared to radiation with vehicle or no treatment (Figure 2G). To address concerns that differences in tumor quantity may alter survival, tumor burden for each brain was assessed.

Average tumor burden for untreated tumors was significantly higher as compared to the mice given temozolomide and radiation (Figure 2H).

The tumor response of QKO tumors after treatment with radiation (+/- temozolomide) mimics the favorable response that is reported in human trials. This validation of QKO tumor response makes the model ideal for testing novel preclinical therapeutics. All of the experiments presented in this figure were performed by Amanda (Mandi) Stang.

PI3K pathway inhibition in QKO tumors

The PI3K pathway is mutationally activated in 88% of HGGs, making inhibition of this pathway an attractive target for HGG therapeutics (Cancer Genome Atlas Research, 2008). Pten, a negative regulator of PI3K, is conditionally deleted in the QKO model resulting in activation of the PI3K pathway in tumors (Fig. 1K, L). We next attempted to inhibit the PI3K pathway in QKO tumors by administering the mTOR inhibitor rapamycin as well as the pan PI3K inhibitor PX-866.

Mice were observed daily for signs of illness. Morbid mice received rapamycin, PX-866, or the combination once daily for five days and were subsequently sacrificed.

To determine PI3K pathway inhibition, western Blot analysis was performed on tumor as well as adjacent normal brain tissue (Figure 3A). Western Blot quantifications indicate pAKT

(Ser473) is significantly decreased in rapamycin treated tumor as compared to control (Figure

3B). Additionally, combination therapy significantly decreases pAKT compared to control (Figure

3A,B). S6 phoshorylation is decreased in both monotherapies as well as the combination when compared to vehicle control tumors (Figure 3A,C). Additionally, the combination therapy significantly decreases pS6 when compared to rapamycin (Figure 3A,C). P4E-BP1 does not

76 significantly change in any treatment regimen when compared to vehicle control tumors (Figure

3A,D).

IHC confirmed a decrease in pAKT and pS6 in treated tumors when compared to control tumors (Figure 3E,F). Moreover, pS6 is decreased in combination therapy when compared to either rapamycin or PX-866 administered alone (Figures 3F). These data indicate both drugs inhibit the PI3K pathway and suggest the combination increases PI3K pathway inhibition as compared to either rapamycin or PX-866 alone.

PI3K inhibition yields variable tumor responses

After confirmation that rapamycin and PX-866 can biochemically alter tumors (Figure 3,

4A,B), we next assessed if there is a biological response in QKO tumors through assessment of proliferation and apoptosis. We observed a great deal of variability in responses. Tumors ranged from <10% KI67 positive cells to >70% positivity (Figure 4C,D). Tumors ranged from 0% positive cleaved Caspase-3 to >80% (Figure 4E,F). Percent positive cells for both KI67 and cleaved

Caspase-3 were plotted to illustrate the variance after identical treatment (Figure 4G, H). Clinical trials utilizing PI3K inhibition have reported varied responses as assessed by survival (Table 1)

(Baselga et al., 2010; Bedard et al., 2015; Bowles et al., 2013; Bowles et al., 2014; Brown et al.,

2015; Hong et al., 2012; Jimeno et al., 2009; Matulonis et al., 2015; Pitz et al., 2015; Rodon et al., 2014; Shapiro et al., 2014; Smith et al., 2014; Soria et al., 2015; Tolaney et al., 2015).

Proliferation marker, KI67, positively correlates with GSX2 and negatively correlates with

Sox 9

Next, we wanted to understand the variability seen in the QKO model after PI3K pathway inhibition. The cancer stem cell hypothesis is based upon the observation that many tumors consist of heterogeneous cell populations of which a subset retains characteristics of stem cells, notably self-renewal and the ability to differentiate into multiple cell lineages (Fiala, 1968; Reya,

Morrison, Clarke, & Weissman, 2001). When isolated, these cells can regenerate the original tumor heterogeneity upon orthotopic implantation into mice. Furthermore, tumor stem cells are often resistant to chemotherapeutics and radiation and are therefore thought to be responsible for treatment failure and recurrent disease (Supplemental Figure 2). To test the theory that this population of cells could contribute to the variability observed in response to treatment in the QKO model, we generated a panel of IHC biomarkers which not only designate progenitor cells but also define where in the differentiation process these cells are located (Table 2). These markers were compared to KI67 by quantification. A total of 500 cells were counted for each marker for each individual tumor. Analysis identified that there was a positive correlation between KI67 and

GSX2 (Figure 5A-E, Table 3). GSX2 is considered a marker of slowly proliferating cells of the subventricular zone. Additionally, analysis shows that there is a negative correlation between KI67 and Sox9 (Figure 5A-C,F,G, Table 3). Sox9 is considered a marker for both oligodendrocyte progenitors and astrocytes. Collectively, these IHCs suggests that tumors with a higher stem cell population are less responsive to PI3K pathway inhibition.

Apoptosis marker, cleaved caspase-3, negatively correlates with Sox10

Using the same IHCs previously generated, we utilized the apoptosis marker cleaved

Caspase-3 to identify if any additional biomarkers would correlate to response. 500 cells were counted for each tumor for each marker of interest. Analysis reveals that cleaved Caspase-3 negatively correlates with oligodendrocyte marker Sox10 (Figure 6A-E, Table 4).

Discussion

The QKO model mimics the histology and genetics of human HGGs. These tumors respond to therapy given to patients with HGG in a similar manner to the responses noted in human HGGs. These traits collectively enable us to study aspects of HGG biology which make the disease difficult for clinicians to treat including tumor heterogeneity, invasiveness and treatment resistance (Marusyk & Polyak, 2010; Navin et al., 2010). Another important aspect of the QKO model is that the tumors generated characterize an underrepresented gene expression subtype, mesenchymal (Simeonova & Huillard, 2014). Friedmann-Morvinski, et al have described a mesenchymal HGG model, however; this model demonstrates dedifferentiation of mature cells into progenitor cells (Friedmann-Morvinski, Bhargava, Gupta, Verma, & Subramaniam, 2014;

Friedmann-Morvinski et al., 2012). These molecular events are present in only a small population of gliomas. Song, et al have described an additional mesenchymal HGG model which is driven by KRAS dysregulation (Song et al., 2013). KRAS is often hyperactivated in GBMs (Guha,

Feldkamp, Lau, Boss, & Pawson, 1997). This model has been utilized in order to understand tumor initiating events. To our knowledge, this model has not been validated for preclinical testing. The QKO mouse is therefore a novel mouse model of HGG that encompasses many features that are useful for preclinical testing: tumors are driven by mutations relevant to human

HGG, the mesenchymal gene expression subgroup is well represented, animals respond in a similar manner to standard treatment options compared to patients and tumor onset is rapid with complete penetrance of the phenotype.

Although the QKO model offers many positive attributes for studying HGG biology, we find interpretation of survival study data difficult due to the multiple tumors generated in most QKO brains. Future studies would benefit from decreasing the tamoxifen administered to QKO mice in order to decrease the number of tumors which are present in each brain. Preliminary data performed suggests we are able to lessen tumor quantity, while increasing time to moribundity by

80 only a small amount with this approach. A decrease in tumor quantity will make this model increasingly valuable for survival study analysis, which may offer a model more recapitulative of human clinical trials. Additionally, MSCV-Cre has been utilized to generate HGG models and labels cells sparsely. This may be an alternative approach to decrease tumor burden if tamoxifen reduction is unsuccessful (Xiao et al., 2005).

Clinical trials which have addressed the effects of PI3K inhibition on tumors (both brain tumors and other solid tumors) have reported varied responses to PI3K treatment

(clinicaltrials.gov) (Table 1). Trials with available tumor material have attempted to identify a prognostic marker (examining components of the PI3K pathway) for the patient population that would benefit from PI3K inhibitor use but have not yet identified such a marker. Proliferation and apoptosis assessment in QKO tumors also show a high degree of variability. Here, we begin but do not definitively identify such a prognostic marker in the QKO model.

Examination of HGG stem and progenitor components has provided insight into a new potential approach for prognostic markers for PI3K inhibitor use. GSX2 is known to mark slowly proliferating cells of the subventricular zone and has been shown to strongly correlate with KI67 staining after PI3K pathway inhibition in QKO tumors (Lopez-Juarez et al., 2013). Additionally,

KI67 negatively correlates with Sox9, which is a marker of astrocyte or oligodendrocyte progenitors (Scott et al., 2010). However, Sox9 identifies a more differentiated cell population compared to GSX2. Cleaved Caspase-3 negatively correlates with Sox10, which is an oligodendrocyte marker (Pozniak et al., 2010). It too represents a more differentiated cell lineage as compared to GSX2. These data suggest that tumors comprised of a more differentiated lineage of cells may respond more favorably to PI3K inhibitor use. QKO-derived tumor spheres show little to no response when subjected to PI3K inhibition (data not shown), further evidence which suggests differentiation of QKO tumors may play a vital component in HGG tumor response after

PI3K inhibitor use.

Further analysis of responsive and non-responsive tumors needs to be conducted to definitively understand the mechanism behind these varied tumor responses. RNAseq analysis will not only examine if there is any potential correlation between tumor response and gene expression subtype, it will enable a more comprehensive analysis of the stem and progenitor components of these tumors.

Another important way to assess responsiveness would be to perform a survival study in which mice are dosed daily until they display signs of moribundity. At this time, mice would be sacrificed and the tumors would be subjected to RNA-seq. Preliminary work has shown that lower doses of tamoxifen yield smaller quantity of tumors (data not shown). A smaller amount of tumors would be necessary in order to be able to properly assess this data as we have seen different tumor responses within the same QKO brain.

An additional assessment which could be easily performed in order to predict tumor response is to correlate location in the brain with tumor response. We note spontaneous tumor development throughout the brain. It is plausible that the location of the tumor may also play a role in responsiveness.

Sequencing analysis of QKO tumors has shown that multiple developmental pathways are also dysregulated in tumors. This could provide rationale for combination therapies in the QKO model which inhibit developmental pathways and potentially drive tumors to a more differentiated lineage followed by PI3K inhibitor treatment. For example, the Notch pathway has been shown to be upregulated in the proneural subset of tumors.

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Figures and Tables

Figure 1: QKO tumors closely mimic human HGGs. (A) Coronal cut of QKO brain with hematoxylin and eosin staining. (B) Sagittal cut of QKO brain with hematoxylin and eosin staining. (C-F) High powered histology of QKO tumors: (C) Anaplastic astrocytoma (D)

Pseudopallisading necrosis (nec) seen in glioblastoma multiform (E) Leptomeningeal disease, * represents the plial membrane. (F) Areas of perivascular spread from a larger tumor. * represents a blood vessel. (G) Gene expression data obtained from our GEMM: Tumors were classified by genetic signature, pictured at the top. Green is proliferative (8% of tumors). Red is

92 mesenchymal (69% of tumors). Purple is proneural (23% of tumors). A total of 39 tumors are represented. (H) Microassay-based Comparative Genomic Hybridization (aCGH) of QKO tumors. Global copy number aberrations are represented by boxes. (I) Kaplan-Meier plot displays the survival curve for QKOs (green), as well as the 2 preceding models. (J-N)

Common immunohistological characteristics of QKO tumors. (J) QKO tumor stained for KI67

(VIP staining with methyl green counterstain). (K) AKT phosphorylation (DAB with hematoxylin counterstain). (L) S6 ribosomal protein phosphorylation (DAB with hematoxylin counterstain).

(M) GFAP (DAB with hematoxylin counterstain). (N) PTEN (DAB with hematoxylin counterstain).

Figure 2: QKO tumors respond similarly to standards of care. (A) Timeline of experimental procedure. (B) Hematoxylin and eosin staining shows histological changes in cellular morphology after radiation, temozolomide, or a combination therapy. (C) Proliferation, as assessed by KI67, is decreased in tumors after radiation or combination therapy. (D) Cellular death, as assessed by Cleaved Caspase 3, is low in all tumors, regardless of therapy. (E)

Quantification of KI67 shows a significant decrease in positive cells after combination therapy as compared to untreated tumors. *Statistics not included-data collected by Amanda Stang prior to

Chow lab shut down. (F) There is no change in cleaved caspase 3. *Statistics not included-data collected by Mandi Smith prior to Chow lab shut down. (G) Kaplan-Meier survival curve for each treatment regimen. *Statistics not included-data collected by Amanda Smith prior to Chow lab shut down. (H) Average tumor burden was assessed in each animal at study end.

Combination therapy yields a significant decrease as compared to untreated controls. *Statistics not included-data collected by Amanda Stang prior to Chow lab shut down.

Figure 3: Short-term treatment reduces PI3K signaling in QKO tumors. Tumor samples were collected after 5 days of treatment and subjected to western blot and/or immunohistochemistry. (A) Representative western blots after 5 days of treatment. (B) One- way ANOVA with Tukey’s multiple comparisons: p=0.0031, F(3,14)=0.73, *(Vehicle and

Rapamycin)=0.0148, *(Vehicle and Combination)=0.013, n=5 tumors. (C) One-way ANOVA with

Tukey’s multiple comparisons: p=0.7438, F(3,14)=0.24. (D) One-way ANOVA with Tukey’s multiple comparisons: p=0.5869, F(3,14)=0.96. (E) Immunohistochemical Evaluation of AKT phosphorylation in tumors after 5 days of treatment. (F) Immunohistochemical evaluation of S6

96 ribosomal protein phosphorylation in tumors after 5 days of treatment. All western blots were performed by Dr. Ralph Salloum, MD.

Figure 4: Tumor response is variable in QKO tumors subjected to a combination of PI3K and mTOR inhibition. (A,B) Rapamycin and PX-866 combination therapy yields a consistent decrease in AKT phosphorylation. (C,D) Tumors show high variability in KI67 after treatment.

(E,F) Tumors are also highly variable in cleaved caspase-3 post treatment. (G) Classification of

KI67 percent positive cells by percent positive show each treatment regimen is represented in each percent classification. One way ANOVA with Tukey’s multiple comparison tests: p=0.8685,

F(3,15)= 0.35; n=5 tumors per group. (H) Classification of cleaved caspase-3 are distributed from 0% to >80%. One way ANOVA with Tukey’s multiple comparison tests: p=0.8389,

F(3,14)=0.12; n=5 tumors per group.

Figure 5: KI67 positivity correlates with GSX2 and negatively correlates with Sox9 in immunohistochemical staining. (A) Tumors were quantified for KI67, GSX2, and Sox9 positive cells. Tumors were then plotted for percent positive cells to assess GSX2 and Sox9 and compare to KI67. (B,C) Representative KI67 immunohistological staining illustrate both high positive cells (B) and low positive cells (C) after daily treatment with rapamycin and PX-866.

(D,E) The same tumors assessed in B and C after immunohistological staining for GSX2. (F,G)

The same tumors assessed in B and C after immunohistoligical staining for Sox9.

Figure 6: Cleaved caspase-3 negatively correlates with Sox10. (A) Tumors were quantified for percent of cleaved caspase-3 and Sox10 positive cells. Tumors were then plotted for percent positive cells to assess potential correlation between apoptosis and Sox10. (B,C)

Representative cleaved caspase-3 immunohistochemical staining illustrate both high positive cells (B) and low positive cells (C). (D,E) The same tumors represented in B and C were also stained for Sox10.

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Supplemental Figure 1: Tumors at 6 weeks post Tamoxifen. (A) Low power hematoxylin and eosin staining of QKO brain, sacrificed at 6 weeks after Tamoxifen (B) High power hematoxylin and eosin staining of the same tumor (C) KI67 immunohistochemistry staining of the same tumor is dark purple (VIP), counterstained with methyl green. (D) Quantification of tumors by location at

6 and 8 weeks post tamoxifen. (n=7).

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Supplemental Figure 2: Tumor stem cell properties of HGGs. (A) Diagram of tumor reimplant procedure. Tumors were removed at moribundity, placed into tumor sphere cell culture, and reimplanted into mice. (B,C) Histological properties of QKO tumors (B) are retained in tumors derived from the reimplant (C). (D) Kaplan-Meier plot of mice implanted with the same tumor (derived from QKO mouse 3936) which had been passed in tumor sphere culture conditions either 3 or 5 passes shows time to moribundity is increased when tumors are maintained in culture for longer periods of time. (Kaplan-Meier survival curve comparison,

Mantel-Cox test: p=0.0246; n=3 per group). (E) Kaplan-Meier plot of mice implanted with the same tumor (derived from QKO mouse 3932) which had been passed in tumor sphere culture conditions either 2 or 4 passes shows time to moribundity is increased when tumors are maintained in culture for longer periods of time. (Kaplan-Meier survival curve comparison,

Mantel-Cox test: **p=0.0339; n=3 per group). Tumor sphere cultures were maintained by Dr.

Annmarie Ramkissoon.

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ANTIBODY MANUFACTURER ID KI67 Cell Signaling 12202S Activated Caspase-3 BD Biosciences 559565 Sox2 Seven Hills WRAB-SOX2 Nestin Millipore MAB353 GFAP Sigma G3893 GSX2 kind give from K. Campbell at Torreson et al., 2000 Cincinnati Children’s Hospital Medical Center MASH1 BD Biosciences 556604 DCX Millipore AB2253 Olig2 Millipore AB9610 Sox9 Santa Cruz SC-20095 Sox10 Santa Cruz sc-17342 CD44 eBioscience 14-0441-85 NICD Cell Signaling 2421S pAKT Cell Signaling 9271 pS6 Cell Signaling 2211 AKT Cell Signaling 4685 S6 Cell Signaling 2217 p-4E-BP1 Cell Signaling 9459 β-actin Sigma 5441

Supplemental Table 1: Antibodies used for all IHC experiments.

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Table 1: Completed clinical trials for solid tumors which utilize PI3K inhibition as well as the reported response. (Brown et al., 2015; Hong et al., 2012; Jimeno et al., 2009; Matulonis et al.,

2015; Pitz et al., 2015; Rodon et al., 2014; Shapiro et al., 2014; Smith et al., 2014; Soria et al.,

2015; Tolaney et al., 2015).

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IHC Antibody Cells Associated Antibody Sox2 Subventricular Zone (postnatal stem cells) Nestin Neural Stem Cells GFAP Characteristic of differentiating and mature astrocytes, stem cell marker of type B cells GSX2 Slow proliferating cells (type B) of the subventricular zone MASH1 Slow proliferating cells (type B) and transit amplifying progenitors (type C) of the subventricular zone Sp8 Neuroblasts of the subventricular zone DCX Olig2 Oligodendrocyte lineage cells in the ventricular zone, proneural HGG histological marker Sox9 Astrocytes, oligodendrocyte progenitors Sox10 Oligodendrocyte progenitors CD44 Cancer stem cell marker, astrocyte-lineage cells, mesenchymal HGG histological marker NICD Notch Intracellular Domain

Table 2: IHC markers for the identification of progenitor cells as well as more differentiated cells within QKO tumors.

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Marker Tukey’s Multiple P-Value Comparison Test Value GSX2** 1.889 0.0019 Sox9** -1.944 0.0011

Table 3: IHC markers which correlate to KI67.

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Marker Tukey’s Multiple P-Value Comparison Test Value Sox10* -1.556 0.0261

Table 4: IHC markers which correlate to cleaved Caspase-3.

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Chapter 3: Intracranial patient-derived xenograft from a pediatric glioblastoma retains

molecular characteristics and growth pathways

Angela White, Annmarie Ramkissoon, Amanda Stang, and Lionel L.M. Chow

My primary contribution was data generation and analysis in Figures 2-4.

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Abstract

Pediatric high-grade glioma (pHGG) can be characterized by both overlapping as well as unique mutations compared to adult HGG. Model systems which recapitulate pHGG are rare, contributing to our lack of understanding about these tumors as well as our lack of efficacious treatment. Here, we established an orthotopic patient-derived xenograft model from a pHGG which was resected from a 12-year-old patient with recurrent disease. Additionally, we assessed a novel therapeutic strategy based on tumor genetic analysis, which was not utilized in the donating patient.

Our model may provide a unique opportunity to study a disease considered difficult to replicate in animals models, pHGG.

109

Introduction

Pediatric high-grade glioma (pHGG) is a devastating disease with poor survival and a high mortality rate (Broniscer, 2006; Broniscer & Gajjar, 2004; Cohen et al., 2011; Finlay & Zacharoulis,

2005). There are both recurring and unique mutations found in these tumors, making assessment and treatment of these tumors a challenge. It is well understood that the driving mutations in pHGG as compared to adult HGG (aHGG) are varied. For example, epidermal growth factor receptor (EGFR) is known to be the most commonly dysregulated receptor tyrosine kinase in aHGG (Cancer Genome Atlas Research, 2008; Network, 2013; Parsons et al., 2008). Platelet- derived growth factor receptor α (PDGFRA) has been identified in multiple subsets of pHGG to be more aberrantly regulated (Bax et al., 2010; Paugh et al., 2010). (There are instances of EGFR mutations in the pediatric population but is reported at a much lower incidence) (Gilbertson et al.,

2003). The genetic differences between adult and pediatric gliomas illustrate why each of these two tumors need their own mouse models for preclinical experiments.

The focus of this thesis overall is PTEN deficiency. PTEN is one of the genes which is known to be regulated differently in both adult and pediatric populations of HGG. As discussed in the introduction of this thesis, PTEN is located on chromosome 10q. In aHGG, loss of heterozygosity (LOH) of chromosome 10q is reported to be approximately 80%. In pHGG, this

LOH is 30%. In aHGG, mutations of PTEN are reported 25%-40% of the time and this number decreases to less than 15% in hHGG (Bax et al., 2010; Brennan et al., 2013, 2014; Pollack et al.,

2006). Additional mutations that create an environment of overactive PI3K signing include mutations of PIK3CA which encodes for the PI3K catalytic isoform p110α and PIK3R1 which encodes for the PI3K regulatory subunit, p85 (Brennan et al., 2013).

As previously discussed in the introduction, PTEN plays many important roles and these roles are context dependent; therefore, PTEN contributions in tumorigenesis are likely to be highly

110 context dependent. Even in similar cell types, additional mutations within an individual tumor are likely to change PTEN’s contributions to tumorigenesis.

Although there are abundant animal models which mimic adult HGG, there are very few established pHGG animal models (Chen, McKay, & Parada, 2012; Coutinho de Souza et al.,

2015; Fomchenko & Holland, 2006; Holland, 2001; Misuraca, Cordero, & Becher, 2015). Hurdles that limit pHGG model establishment include the smaller patient population, difficulties in establishing cell cultures, and tumors which do not serially engraft.

Pediatric models are greatly needed in order to understand the mechanisms that drive pediatric gliomagenesis as well as to test and improve therapeutic strategies. Here we present a pediatric primary orthotopic model from a patient-derived tumor which contains many common mutations seen in pHGG. Additionally, we present pre-clinical testing with this established model.

This sample represents a patient population which has undergone one round of therapy and is reoccurring. Samples which are naive to therapy as well as recurrent disease are both needed as they represent two distinct patient populations, each of which need to be studied in animal models in order to formulate better therapeutic strategies.

111

Materials and Methods

Tumor Implant and Collection of Samples

Tumors were surgically resected and a portion donated with parental consent, for laboratory research. The tumor was dissociated and prepared for injection immediately upon arrival by Dr. Annmarie Ramkissoon, PhD. In order to lessen the time from surgical removal to injection, cells were not counted. The prepared tumor sample was directly implanted into the brains of NOD SCID Gamma (NSG) mice (Jackson Laboratory). NSG mice have mutations that create severe immunodeficiency, specifically IL2 receptor common gamma chain (IL2rg) and severe combined immune deficiency (scid). The IL2rg mutations leads to natural killer (NK) cell deficiency. The scid mutation leads to B and T cell deficiency. This immunodeficiency allows these mice to be successfully engrafted with human tumor material, making this a favorable model for patient-derived xenografts (Ito et al., 2002).

The injection site was 2 mm right, 1 mm anterior to the bregma suture. The needle of the

Hamilton syringe is lowered 3.5 mm into the brain. 1 uL of the prepared tumor sample was injected each minute for a total of 3 minutes. Mice were monitored daily for the first week after surgery for recovery. Mice were then assessed 3 times per week for signs of illness associated with tumor.

Mice were euthanized at morbidity or after completion of therapy.

Mice were anesthetized with 2,2,2-Tribromoethanol (Sigma-Aldrich) and perfused with 1x phosphate buffered saline (PBS) for tissue exsanguination. Brains were removed and immersed in 4% paraformaldehyde (4% PFA) in 1x PBS for 24 hours. Frozen tissue was stored at -80C, for protein or nucleic acid preparations. PFA preserved tissue was processed and embedded in paraffin. 5 um sections were cut for histology and IHC.

112

Drug Formulation

Rapamycin (LC-Laboratories) stock was prepared in DMSO at a concentration of

50mg/ml. Aliquots were prepared weekly and stored at -20C. Aliquots were thawed daily and diluted with 5.2% Tween 80 in ddH2O to a concentration of 2.5mg/ml prior to administration.

BKM120 stocks were made at 50mg/ml in N-methyl-2-pyrrolidone and stored at -20C. Stocks were diluted daily to a concentration of dosage of 5 mg/ml in PEG 300.

Histology and Immunohistochemistry

Sections were deparaffinized with xylene, passed through graded alcohols, and stained with Hematoxylin and eosin stain (H&E) reagents. Immunohistochemistry (IHC) sections were similarly deparaffinized and passed through graded alcohols to water. Antigen retrieval was achieved by immersing slides in citrate buffer and boiling slides. Next, slides are immersed in 1%

H2O2 for 30 minutes and subsequently blocked for minimum of an hour in 10% goat serum. Table

1 lists the antibodies used for IHC.

113

Results

Genetic Landscape of Contributing Tumor and Serial Transplants

This tumor originated from a 12-year-old male who underwent a third surgical resection for recurrent glioblastoma (GBM). He had previously received focal radiation therapy, temozolomide and bevacizumab at diagnosis, and combinations of vorinostat, temozolomide, etoposide, bevacizumab and irinotecan after his first recurrence.

Tumor samples from the first two resections were not sent for molecular profiling however the current tumor sample was analyzed by FoundationOne™ sequencing and found to contain several driver mutations known to contribute to gliomagenesis through processes such as proliferation, cell cycle deregulation, and stem cell maintenance (Table 2) (Bjerke et al., 2013;

Duerr et al., 1998; Endersby & Baker, 2008; Gallia et al., 2006; Hartmann, Bartels, Gehlhaar,

Holtkamp, & von Deimling, 2005; Koul, 2008; Ohgaki, 2005; Pandey, Bhaskara, & Babu, 2016;

Schwartzentruber et al., 2012; Vogelstein, Lane, & Levine, 2000; Westhoff et al., 2014; Yang et al., 2010). To confirm these mutations were present in each xenograft, DNA was extracted from the tumors from each serial engraftment and subjected to Cincinnati Children’s Hospital Medical

Center’s (CCHMC) Focus Cancer Panel (FCP). FCP is able to detect single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs), and insertions/deletions

(INDELs) within targeted regions of oncogenes or tumor suppressor genes deemed clinically relevant. A set of these mutations were confirmed in both the patient-derived sample as well as each serial transplant (Table 3). Three of the 4 mutations maintain a consistent percentage from the donating tumor and through each passage. PIK3CA increased from 46% in the donating tumor to approximately double in the first passage. The high percentage was maintained throughout each passage. Mutation landscape can change during or after chemotherapy so it is possible the process of removing the tumor as well as the process of preparing the tumor for implantation induces a response which alters aspects of the genetic makeup of the donating

114 tumor. Additionally, it’s possible that the certain cells in the heterogeneous population die faster than other populations and the mutations could either increase or decrease as a proportion of what cells are living.

H3F3A is one of the three genes that encodes histone variant H3.3, which is known to be important in chromatin remodeling (Gessi et al., 2013; Plass et al., 2013). G34R/V mutations are known to be present in approximately 15% of pediatric GBM. This mutation is not present in adult

GBMs (Bjerke et al., 2013; Fontebasso et al., 2014; Plass et al., 2013; Schwartzentruber et al.,

2012).

FoundationOne™ sequencing of the patient sample confirmed that the G34R mutation was present (Table 2). Two DNA samples from each of the three serial implants along with the patient sample were amplified via PCR for the H3F3A region of interest and sent to the CCHMC

DNA Sequencing and Genotyping Core for sequencing. The mutation was confirmed in the patient sample and each engraftment (Figure 1).

These data confirm that each serial transplant of the patient-derived tumor retains the mutation profile of the original tumor.

Histological and Immunohistochemical Characteristics

Once the mutations were confirmed in both the patient sample and the serial transplants, we wanted to assess the histological and immunohistochemical characteristics of both the patient tumor and the subsequent transplants.

Histological examination show that both the cellular and stromal components of each serial passage of the tumors mimic that of the original tumor (Figure 2A,B). Glial fibrillary acidic protein (GFAP) is a protein which forms intermediate filaments in astroglial cells and is thus used

115 as a marker of mature astrocytes (Figure 2C). P53 expression is high in both the donating tumor as well as the serial transplants. There was a mutation in TP53 (Table 2). High expression of p53 is associated a poor prognosis in pediatric gliomas (Figure 2D) (Pollack et al., 2002). PTEN mutations, as previously reviewed, are common in aHGG but less frequent in pHGG. Deficiency is noted in Figure 2E.

To assess hyperactivation of the PI3K pathway, we next assessed both AKT phosphorylation and S6 phosphorylation. Consistently, in the donating tumor and serial passages, we see high levels pS6 and pAKT but it is difficult to speculate on the quantity of elevation as there is no negative control (Figure 2F,G).

Proliferation, as assessed by KI67, is consistent in each passage of tumor material

(Figure 2H). Similarly, apoptosis, which is seen in limited percentages, is preserved in both the original tumor and each passage (Figure 2I). Finally, anti-human nucleoli was assessed in order to confirm the principle component of the tumor remains human. As seen in Figure 2J, the cellular component of each tumor stains positive (brown) while neighboring tissue from the mouse is negative (blue).

Tumor Reponses to PI3K Pathway Inhibition

Foundation One™ sequencing demonstrated that multiple mutations affecting pathways that can be targeted by specific small-molecule inhibitors were present in this tumor. This patient did not receive such therapeutics. Several mutations in this tumor activate the PI3K pathway.

PIK3CA encodes for the p110α catalytic isoform of PI3K. Mutations in PIK3CA are common in cancer, and specifically gliomas. Mutations in PI3K isoforms ultimately lead to an increase in the downstream signaling in the PI3K pathway. There is also a driving mutation in PTEN in the tumor.

As extensively discussed in the introduction of this thesis, PTEN is a commonly mutated gene in

116 many different forms of cancer, including gliomas. PTEN mutations yield a loss of PTEN proteins which negatively regulate the PI3K pathway, thus increased signaling downstream of PI3K is one of the results of PTEN mutations. In adults, mutations in PTEN are associated with poor prognosis

(Knobbe, Merlo, & Reifenberger, 2002). In pediatric diffuse astrocytic gliomas, PTEN mutations were considered a marker of poor prognosis (Knobbe et al., 2002).

These mutations were preserved in the xenograft model (Table 1). We therefore decided to test the effects of PI3K inhibitors on the patient-derived xenograft. BKM120 is a pan-PI3K inhibitor and rapamycin inhibits mTOR (Figure 3). Mice from pass 2 were implanted and observed daily for signs of illness (weight loss, dehydration, etc). We administered either the

PI3K inhibitor BKM120 or the mTOR inhibitor, rapamycin daily for five days at the onset of first symptoms of tumor burden.

First, we assessed if PI3K pathway signaling is decreased after either BKM120 or

Rapamycin. Immunohistochemistry of both pAKT and pS6 confirms these compounds inhibit the pathway (Figure 4 A, B). Next, we assessed proliferation and apoptosis. KI67 decreased from

55.6% after vehicle administration to 30.8% and 15.4% after rapamycin or BKM120 administration, respectively (Figure 4C). No changes in cleaved Caspase-3 were detected

(Figure 4D). These data suggest that there may be a therapeutic benefit in using small-molecule inhibitors to decrease aberrantly regulated pathways in gliomagenesis. It is imperative to note that, due to the low number of mice on this study, no significant conclusions have been made.

117

Discussion

Radiation and temozolomide are the current pHGG standard of care and often only improve survival by a few months (Kline, 2017). To date, molecularly targeted agents have been used most successfully in situations where specific mutations are identified that respond to the drug. There are fewer examples where inhibition of the pathway deregulated by mutation can be beneficial (Hoelder, Clarke, & Workman, 2012). Furthermore, tumors that are driven by multiple pathways are likely to require combined pathway inhibition. It is important to define these requirements in pre-clinical studies so that appropriate clinical trials can be designed that identify molecular biomarkers to stratify patients most likely to benefit from treatment. Here, we developed and defined a model which represents a disease often considered difficult to accurately replicate in animal models, pHGG.

At the time of biopsy, multiple labs attempted to culture this tumor sample with no success.

Additionally, the first and second serial transplant failed to thrive in culture. Utilizing orthotopic transplantation may provide the best approach to increase the quantity of pediatric models available. Additionally, we have observed that sample collections at the time of biopsy tend to thrive as compared to autopsy samples. These observations may aid in creating additional pHGG models.

Finally, pertinent to this thesis, we assessed PTEN deficiency in this pHGG model. The preliminary work here used a therapeutic approach which inhibited the PI3K pathway because there are two mutations in this tumor which may yield increases of PI3K signaling. This ultimately increases cell size, cell cycle progression, and proliferation. These results are not statistically powered so it is difficult to make statements about the effects of using either a PI3K inhibitor or an mTOR inhibitor; however, the preliminary results suggests both BKM120 and Rapamycin decrease cell proliferation which suggest potential therapeutic utility.

118

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Figures and tables

Figure 1: Conservation of H3F3A G34R mutation in patient-derived tumors. DNA was extracted from the patient tumor and from patient-derived xenograft (PDX) tumors. The H3F3A gene was amplified by PCR and sequenced by Sanger sequencing using an ABI Prism 3730xl

DNA Analyzer. Results were analyzed using SnapGene 3.1 software. The amino acid position and single-letter code is indicated above. Histogram colors are green: A; blue: C; black: G; red:

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Figure 2: Histology of pGBM PDX. The histological features of the patient tumor were preserved in multiple generations of the PDX including the appearance of the tumor under H&E

125 staining (A,B), several pathway markers (C-G), extent of proliferation (H), and apoptosis (I).

Human cells were detected with anti-human nucleoli (J).

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Figure 3: PI3K Pathway and Inhibition. This simplified version of the PI3K pathway shows where the small molecule inhibitors utilized in this work inhibit the pathway. BKM120 is a pan-

PI3K inhibitor. Rapamycin inhibits mTOR.

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Figure 4: Response to PI3K pathway inhibition. PDX mice (Pass 2) showing signs of tumor growth were treated with 25 mg/kg rapamycin ip, 50 mg/kg buparlisb (BKM120) by oral gavage or vehicle daily for five days. The tumor was removed after the last dose and analyzed by immunohistochemistry. PAKT, pS6, and KI67 decrease with either rapamycin or BKM120. N=1 per group.

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ANTIBODY MANUFACTURER ID KI67 Cell Signaling 12202S Activated Caspase-3 BD Biosciences 559565 pAKT Cell Signaling 9271 pS6 Cell Signaling 2211 AKT Cell Signaling 4685 GFAP Sigma G3893 P53 Cell Signaling 2527 PTEN Cell Signaling 9559

Table 1: Antibodies utilized for immunohistochemical staining of both the donating tumor as well as he serial passages.

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MUTATION PATHWAY (OR PROCESS) TARGETED ALTERED THERAPIES KRAS G13D – MAPK pathway MEK Inhibitors subclonal PIK3CA Q546E PI3K pathway PI3K and mTOR inhibitors PTEN K267fs*9 PI3K pathway PI3K and mTOR inhibitors TP53 H193Y, L93fs*30 regulates apoptosis and senescence -- H3F3A G35R Histone H3.3 Epigenetic regulators

Table 2: Driver mutations of HGG identified in this tumor sample with FoundationOne™ sequencing, as well as the pathway or cellular process which is altered.

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MUTATION PATIENT PASS 0 PASS 1 PASS 2 PIK3CA Q546E 46% 100% 51% 100% 99.9% 99.98% 100% PTEN K267fs 90% 93% 94% 96% 94% 95% 97% TP53 H193Y 48% 48% 50% 43% 47% 44% 53% TP53 L93fs 49% 52% 51% 59% 52% 57% 50%

Table 3. Conservation of driver mutations in PDX tumors. DNA was submitted for the Focus

Cancer Panel Test, a CLIA certified next generation sequencing test of 50 genes commonly mutated in pediatric cancers. Allele frequencies of each mutation are reported from the patient and from two independent tumors from each PDX generation.

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Chapter 4: PI3K isoform-selective inhibition in neuron-specific PTEN-deficient mice

rescues molecular defects and reduces epilepsy-associated phenotypes

Presented as published in: White, A. R., Tiwari, D., MacLeod, M. C., Danzer, S. C., & Gross, C. (2020). PI3K isoform-selective inhibition in neuron-specific PTEN-deficient mice rescues molecular defects and reduces epilepsy-associated phenotypes. Neurobiol Dis, 144, 105026. doi:10.1016/j.nbd.2020.105026

My primary contribution was data generation and analysis in Figures 1-7 and Supplemental Figures 1-5

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Abstract

Epilepsy affects all ages, races, genders, and socioeconomic groups. In about one third of patients, epilepsy is uncontrolled with current medications, leaving a vast need for improved therapies. The causes of epilepsy are diverse and not always known but one gene mutated in a small subpopulation of patients is phosphatase and tensin homolog (PTEN). Moreover, focal cortical dysplasia, which constitutes a large fraction of refractory epilepsies, has been associated with signaling defects downstream of PTEN. So far, most preclinical attempts to reverse PTEN deficiency-associated neurological deficits have focused on mTOR, a signaling hub several steps downstream of PTEN. Phosphoinositide 3-kinases (PI3Ks), by contrast, are the direct enzymatic counteractors of PTEN, and thus may be alternative treatment targets.

PI3K activity is mediated by four different PI3K catalytic isoforms. Studies in cancer, where

PTEN is commonly mutated, have demonstrated that inhibition of only one isoform, p110β, reduces progression of PTEN-deficient tumors. Importantly, inhibition of a single PI3K isoform leaves critical functions of general PI3K signaling throughout the body intact. Here, we show that this disease mechanism-targeted strategy borrowed from cancer research rescues or ameliorates neuronal phenotypes in male and female mice with neuron-specific PTEN deficiency. These phenotypes include cell signaling defects, protein synthesis aberrations, seizures, and cortical dysplasia. Of note, p110β is also dysregulated and a promising treatment target for the intellectual disability Fragile X syndrome, pointing towards a shared biological mechanism that is therapeutically targetable in neurodevelopmental disorders of different etiologies. Overall, this work advocates for further assessment of p110β inhibition not only in

PTEN deficiency-associated neurodevelopmental diseases but also other brain disorders characterized by defects in the PI3K/mTOR pathway.

133

Introduction

Several studies have identified an association between mutations in the phosphatase and tensin homologue (PTEN) gene and neurodevelopmental disorders, in particular in individuals with macrocephaly (Butler et al., 2005; Goffin et al., 2001; Kwon et al., 2001; Pinto et al., 2014). A causal relationship between impaired PTEN expression and epilepsy, autistic-like behavior and brain structural abnormalities was confirmed in mouse models with neuronal subset-selective knockout of Pten (Backman et al., 2001; Kwon et al., 2006; Kwon et al., 2001;

Pun et al., 2012). These studies, which used different promoters and approaches to delete Pten in subsets of neurons in the brain have shown that in mice, neuron-specific deletion of Pten consistently leads to spontaneous seizures. Patients with germline mutations of PTEN rarely develop refractory epilepsy (Rademacher and Eickholt, 2019), but somatic mutations in the

PTEN/phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway, which better reflect the brain-specific mouse models, have often been found in patients with focal cortical dysplasia (FCD) (Lim et al., 2015; Lim and Crino, 2013). In addition, increased

PI3K/mTOR signaling has been observed in FCDs (Schick et al., 2006). FCDs are a frequent cause of refractory epilepsy, and thus novel treatments targeting the PTEN/PI3K/mTOR pathway to reduce seizure burden in these disorders are urgently needed.

PTEN’s lipid phosphatase function counteracts the class I PI3K pathway by catalyzing the dephosphorylation of phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3) at the 3’ phosphate of the inositol ring to phosphatidylinositol (4,5)-bisphosphate (PIP2) (Maehama and Dixon,

1998). PTEN, through the regulation of PIP2/PIP3 ratios, can affect cell signaling downstream of

PI3K, including protein kinase B (AKT) and mTOR (Switon et al., 2017). Through the negative control of PI3K, PTEN regulates many cellular functions, such as metabolism, growth, and mitosis (Carnero et al., 2008).

MTOR inhibition has been preclinically assessed as a therapy for autism and epilepsy in

PTEN-deficient mice. The mTOR-inhibitor rapamycin rescues many aberrations seen in neuron-

134 specific PTEN-deficient mice including changes in neuronal structure, brain morphology, and behavioral deficits (Kwon et al., 2003; Zhou et al., 2009). Additionally, long-term treatment with rapamycin decreases the progressive epilepsy in these mice (Sunnen et al., 2011). A recent study has shown that, likewise, mTORC2 inhibition rescues neurological phenotypes in neuron- specific PTEN-deficient mice (Chen et al., 2019), and an mTOR inhibitor is currently being evaluated to treat cognitive deficits in individuals with PTEN mutations (clinicaltrials.gov).

However, mTOR is several molecular steps downstream of PI3K, thus PIP3 accumulation is still expected to occur in the membrane even if mTOR is blocked. PIP3 plays an important role in neuronal polarization as well as axon formation, and thus is central for neuronal function

(Menager et al., 2004). This suggests that therapeutic strategies that target further upstream components of this pathway could be beneficial for neurodevelopmental disorders caused by

PTEN mutations. To test this hypothesis, we investigated an alternative strategy targeting PI3K activity to correct neuronal deficits associated with PTEN deficiency, which could be applicable to other neurodevelopmental disorders with mutations in the PI3K/mTOR pathway.

PTEN mutations also occur frequently in a multitude of cancers, and drug discovery in

PTEN-associated neurological disorders may thus benefit from advances in cancer research.

Studies in PTEN-deficient cancers have demonstrated that inhibition of one of the four class I

PI3K isoforms, p110β, will stop or significantly reduce tumorigenesis (Jia et al., 2008; Wee et al., 2008). This strategy may prove favorable compared with other mTOR or PI3K-modifying strategies, as it targets the direct source of dysregulation, reduces potential side effects of pan

PI3K inhibition, and lessens the chances of reactivation of PI3K signaling in a feedback loop

(Wee et al., 2008). Class 1 PI3K isoforms can have distinct and overlapping functions, and previous research suggests that they signal downstream of specific membrane receptors (Gross and Bassell, 2014; Vanhaesebroeck et al., 2010), but so far, the specific PI3K isoforms and receptors that drive PI3K signaling in PTEN-deficient brain disorders are unknown.

135

P110β is expressed in the brain with a nearly uniform distribution pattern (Lein et al.,

2007). Apart from p110β dysregulation in PTEN-deficient tumors, the inherited intellectual disability Fragile X syndrome (FXS) is associated with overactive p110β (Gross and Bassell,

2012; Gross et al., 2010), and copy number variants in the PIK3CB gene (coding for p110β) were found in patients with autism (Cusco et al., 2009). These studies suggest that p110β dysregulation is a shared molecular defect and treatment target in neurodevelopmental disorders of different etiologies. Our lab has demonstrated that genetic and pharmacologic inhibition of p110β rescue molecular, cellular and behavioral phenotypes in mice with a deletion of Fragile X Mental Retardation Gene 1 (Fmr1), which is silenced in individuals with FXS (Gross et al., 2019; Gross et al., 2015). Most recently, we have used a p110β-selective inhibitor which penetrates the blood-brain barrier, GSK2702926A (GSK6A) and has a similar chemotype as one currently in clinical trials for PTEN-associated cancer (Gross et al., 2019; Mateo et al.,

2017). Based on our promising findings in FXS and published results in PTEN-deficient cancer, we hypothesized that p110β inhibition may ameliorate phenotypes in PTEN mutation-associated neurodevelopmental disorders.

Using mice in which Pten is deleted postnatally in excitatory forebrain neurons

(CamKIIα-cre; Ptenfl/fl) (Chen et al., 2019; McMahon et al., 2012; Sperow et al., 2012), we show that pharmacological inhibition of p110β with GSK6A reduces enhanced PI3K/AKT signaling and protein synthesis rates, and reduces seizure frequency. We also show that in another neuron-specific PTEN-deficient mouse model (Gfap-Cre; Ptenfl/fl) (Kwon et al., 2006; Kwon et al., 2001), p110β inhibition ameliorates signaling defects and cortical dysplasia, and reduces the size of CA1 neuron soma and dentate granule cell soma. This work supports the potential utility of repurposing a therapy developed for PTEN-deficient cancer for a subset of individuals with neurodevelopmental disorders.

136

Methods and Materials

Mice

Floxed Pten mice and CamK2α-Cre mice were obtained from Jackson Laboratory

(B6;129S4-Ptentm1Hwu/J, JAX Mice stock number 006440, RRID:IMSR_JAX:006440; and B6.Cg-

Tg(Camk2a-cre)T29-1Stl/J, JAX Mice stock number 005359, RRID:IMSR_JAX:005359). Pten;

CamKdel (deletion) mice and their littermate controls were generated by crossing male Ptenfl/+;

CamK Cre+ with either female Pten floxed heterozygous or Pten floxed homozygous mice.

Pten; Gfapdel (deletion) mice were generated by breeding Ptenfl/+; Gfap+ (kind gift from Dr.

Matthew C. Weston at the University of Vermont) (Backman et al., 2001; Kwon et al., 2001).

Mice were genotyped via polymerase chain reaction. Littermate controls were either Ptenfl/fl;

CamK Cre- or Pten+/+; CamK Cre+. For macrocephaly assessment, controls were Pten+/+;

Gfap+. Mice were housed at a 14:10 hour light/dark cycle in standard cages with up to 4 animals with food and water ad libitum. The animal protocol was approved by the Institutional

Animal Care and Use Committee of CCHMC and complied with the Guide for the Care and Use of Laboratory Animals.

Dose escalation and protein synthesis studies were performed with mice at the age of 6 to 7 weeks. A total of 25 mice (including 15 females and 10 males) were designated for dose escalation studies. A total of 31 mice were designated for protein synthesis assays, including 16 females and 15 males. Cortical analysis was added after the initial hippocampal data collection resulting in a higher number of mice than n reflected in the figure. Daily intraperitoneal (i.p.) injection experiments were performed on mice at 6 weeks of age. A total of 9 mice (including 4 females and 5 males) were designated for these experiments. Ten male and 17 female mice at the age of 7 weeks were used for nesting assays and analysis of signal transduction. All seizure analysis studies were started when mice were approximately 8 weeks. For EEG experiments, a

137 total of 16 Ptenfl/fl; CamK Cre+ mice completed the entire experiment. Five mice were designated for EEG experiments but died prior to surgery. Thirteen mice underwent EEG electrode implantation but died prior to receiving treatment. Three mice died during the treatment portion of the experiment. A total of 12 littermate controls completed the experiment.

One littermate control died during the experiment due to unknown causes (also see Table 2).

No seizures were noted in any of the control mice, including the one mortality. Two mice from the same litter were excluded due to excessively high seizure burden (>100 seizures per day).

A total of 21 female mice and 28 males were used for the completion of EEG experiments. In assessing mortality, there were no differences in sex distribution (data not shown). Long-term dosing for histological analysis was started when mice were 4 weeks of age. A total of 21 mice were designated for histological analysis, including 10 females and 11 males.

Primary Neuronal Culture

Embryonic cortical and hippocampal neurons were prepared and cultured in 12-well plates as previously described (Muddashetty et al., 2007). Pten and scrambled siRNA were transfected by magnetofection using Neuromag Transfection Reagent (Oz Biosciences, San

Diego, CA) according to the manufacturer’s protocol. Briefly, 1 µg siRNA and 3.5 µl Neuromag beads were added to each well of a 12-well plate 8 days after plating. After 15 minutes of incubation on the magnetic plate, neurons were cultured for 3 days prior to drug treatment and lysis.

Antibodies and siRNAs

The following primary antibodies were utilized from Cell Signaling Technology (Danvers,

MA, USA) for western blotting and immunoprecipitations: CRKL (Cat: 38710,

RRID:AB_2799138), IgG (Cat: 2729, RRID: AB_1031062), AKT (Cat: 2920, RRID:

AB_1147620), pAKT (T308) (Cat: 4056, RRID: AB_331163), pAKT(S473) (Cat: 4060, RRID:

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AB_329825), pS6 (S240/244) (Cat: 5364, RRID: AB_10694233), S6 (Cat:2317, RRID:

AB_2238583), Phospho-p70 S6 Kinase (Cat: 9204, RRID: AB_2265913), pPDK1(Cat:3438,

RRID: AB_2161134), PDK1 (Cat: 5662, RRID: AB_10839264) and PTEN (Cat: 9559, RRID:

AB_390810). The following additional antibodies were used: Tubulin β-3 (BioLegend, San

Diego, CA, Cat: USA-802001, RRID: AB_2564645) and puromycin (The University of Iowa,

Iowa City, Iowa, Cat: USA-PMY2A4-S, RRID: AB_2619605).

SiRNAs were obtained as custom stealth RNAi siRNA from Invitrogen Life Technologies,

Thermo Scientific (Carlsbad, CA, USA), with the following sequences: PTEN siRNA (5’ to 3’):

CAG CCA UCA UCA AAG AGA UCG UUA G, (5’ to 3’): CUA ACG AUC UCU UUG AUG AUG

GCU G, Control (5’ to 3’): CAG ACU AAA CUG AGA GCU AUC CUA G, Control (5’ to 3’) CUA

GGA UAG CUC UCA GUU UAG UCU G.

Immunoprecipitations

Primary cortical neurons, PC3 cells (ATCC, Manassas, VA, USA, Cat: ATCC CRL-1435,

RRID:CVCL_0035) and frozen wild type mouse cortex were lysed and fresh lysates were split at

600 μl between IgG (3 μl) and CRKL (12 μl) antibodies. The lysis buffer was composed of 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton x-100, 50 mM Tris HCl

(pH=7.4), 50 mM NaF, 10 mM Na pyrophosphate, and 10 mM Na beta-glycerol phosphate. The samples were rotated at 4˚C for 2 hours. Protein A sepharose beads (General Electric

Healthcare, Chicago, IL, USA, Cat: GE17-0780-01) were washed with lysis buffer for a total of 3 times. After the 2-hour incubation, 50 μl of beads were added to each sample condition and rotated overnight at 4˚C. The following day, each set of beads were washed 3 times with 250 μl of lysis buffer and samples were subjected to western blotting.

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Protein Synthesis Assay and GSK Dose Escalation

Serial brain slices were prepared in oxygenated cutting solution (75mM sucrose, 25mM glucose, 87mM NaCl, 25mM NaHCO3, 2.48mM KCl, 1.42mM NaH2PO4, 14.96mM NaH2PO4,

14.96mM MgCl2, 511mM Ascorbate). Hippocampus and somatosensory cortex were dissected and incubated in oxygenated artificial cerebrospinal fluid (25mM glucose, 125mM NaCl, 25mM

NaHCO3, 2.5mM KCl, 1.42mM NaH2PO4, and 2.15mM MgCl2) in a 12-well plate for 30 minutes at 31˚C.

For dose escalation studies, increasing doses of GSK6A (0 to 10 μM) were added to the wells and incubated for 30 minutes. Samples were then collected in 1.5 milliliter vials and immediately frozen on dry ice.

For protein synthesis assays, GSK6A (1 μM) or vehicle was added to the wells and incubated for 30 minutes. Puromycin (Life Technology, Waltham, MA, Cat: USA-A1113803) (10 mg/ml) was next added to each well of the plate, except for the “no puromycin” control. Samples were then incubated for 40 minutes and immediately frozen on dry ice.

SDS-PAGE and Western Blot Analysis

Protein concentration was determined using Bio-Rad Protein Assay Dye (Hercules,

California, USA; Cat: 5000006). Samples were mixed with SDS sample buffer and equal amounts of proteins were loaded in duplicate on SDS-PAGE, and transferred to PVDF Transfer

Membrane (Millipore Sigma, Darmstadt, Germany). Membranes were blocked using 5% milk for

1 hour. Antibodies were diluted to the desired concentration in 1% Tween in PBS and incubated overnight at 4˚C. Membranes were then washed and incubated with secondary antibody, either

Rabbit IgG HRP Linked Whole Antibody (Millipore Sigma, Darmstadt, Germany; Cat: GENA934) or Mouse IgG HRP Linked Whole Antibody (Millipore Sigma, Darmstadt, Germany; Cat:

NXA931V). Signals were detected with enhanced chemiluminescence using Pierce ECL Blotting

Substrate (Thermo Scientific, Carlsbad, CA, USA, Cat: 32106). If a second detection was

140 needed, blots were stripped using Restore western Blot Stripping Buffer (Thermo Scientific,

Carlsbad, CA, USA, Cat: 21059), blocked again in 5% milk, and incubated overnight with the desired antibody.

Western Blot Quantification

Specific signals on western blots were quantified densitometrically using NIH ImageJ software (Bethesda, Maryland, USA). Signal intensities of phospho-AKT, phospho-S6K, PTEN, and Puromycin were normalized to AKT or β-Tubulin signal on the same blot. Phospho-PDK1 and phospho-S6 signal intensities were normalized to the signal intensities of total PDK1 and

S6 on the same blot, respectively. The average of the duplicates was counted as one data point.

Drug Formulation and Administration

GSK2702926A was synthesized at GlaxoSmithKline as previously described by Rivero et al (2014). For intraperitoneal (i.p.) dosing, a dose of 5 mg/kg (0.01 g/ml in 10% dimethyl sulfoxide (DMSO) (BioWorld, Dublin, OH, USA)) was formulated fresh daily from frozen stock in DMSO. Vehicle control was 10% DMSO. For oral dosing, GSK6A was formulated at a dose of 5 mg/kg in Jif® peanut butter. Mouse weights were averaged, and peanut butter pellets were formulated and frozen for an average mouse weight of 23 grams. The average of weight for the mice used in this study was 22.28 grams with an SEM of 0.5988. Mice were observed to ensure ingestion of the pellet, which typically occurred within 5 minutes. 10 μM GSK6A stock in DMSO was formulated to a concentration of 1 μM prior to use. BYL719 (Alpelisib)

(VWR International, Radnor, PA, USA) was formulated to a 5 mM stock in DMSO and diluted to 0.05 μM. 2 mM IC87 (IC87114, Millipore Sigma, Darmstadt, Germany) stock was likewise prepared in DMSO and diluted to 2 μM for use.

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Nest Building Behavior

Nesting behavior was assessed as previously described (Deacon, 2006) with mice between 7 and 10 weeks of age. Briefly, nestlets were weighed at 0 and 2 hours after addition to a cage with a single-housed mouse and percent torn was recorded. Some mice were subjected to two nesting behavior assays, one prior to the treatment regimen (Fig. 4A) and one at the end of treatment (Fig. 4B). Both naive and mice with electrode implants were utilized.

Surgical Implantation of Electrodes

Cortical surface electrodes were surgically implanted as previously described (Castro et al., 2012; Tiwari et al., 2019). Briefly, mice were anesthetized in a chamber with 3% isoflurane. Once the mice were confirmed to be at the proper level of anesthesia, the mice were placed on the stereotaxic frame and received maintenance anesthesia at 1% to 2% isoflurane.

Two drill holes were created without penetrating the dura for the placement of surface transmitter electrodes (relative to bregma AP= -2.5 mm, L: 2 mm for both sides). The right electrode was used for signal and the left was used for reference. The transmitter device

(TA11ETAF-10, Data Sciences International (DSI), Saint Paul, MN, USA) was placed in the subcutaneous pocket spanning from the neck to the left flank. The transmitter electrodes were inserted into the burr holes on the dura without injuring it and fixed into place with Ortho Jet

Liquid Powder Kit, (Patterson Dental, Saint Paul, MN, USA). The incision was closed with suture

(Coviden, Dublin, Ireland), GLUture (Zoetis Inc., Kalamazoo, MI, USA) and treated with antibiotic ointment (RARO, Hawthorne, NY, USA).

Seizure Assessment

Approximately five days after cortical surface electrode implant, seizure presence was assessed through EEG recordings using a wireless EEG system from Data Sciences

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International. NeuroScoreTM DSI (St. Paul, MN) software was used for seizure assessment. A seizure was defined as sudden onset of high frequency and amplitude activity (>2x baseline) and a duration greater than ten seconds. Five days of EEG were recorded after seizures had been confirmed to establish baseline seizure frequency before treatment started. The seizures of this model are well-defined, but video monitoring was available if additional confirmation was needed.

EEG Spike and Power Analysis

EEG spikes and power were analyzed during interictal times, at least one hour before or after the last seizure. EEG spikes were detected with a spike detector module in NeuroScore™ using the built-in dynamic threshold protocol to accommodate variations in EEG signal between mice. A spike was defined as having a duration of 5-80ms, with a dynamic threshold ratio of 3-6 and a minimum amplitude of 100 µV. Spikes were analyzed for 2-hour periods during the day

(12-6pm) and the night (12-6am), and grooming and other signal artifacts were removed. For power analysis, the raw EEG signal was exported into 10s epochs to generate accumulative power bands from 2 hours of recording during the day (12-6pm) and night (12-6am) and subjected to Fast Fourier Transformation to generate power bands. The EEG signal was split into power bands of various frequencies (shown here: gamma, 24-80 Hz) and normalized to total power to account for baseline differences in EEG signal.

Brain Morphology Assessment

Gfap-Cre; Ptenfl/fl (henceforth, Pten; Gfapdel) mice and littermate controls (Pten+/+; Gfap

Cre+) were dosed intraperitoneally daily with either GSK6A or vehicle for four weeks. After the final dose, the brain was dissected and preserved in 4% paraformaldehyde. A few brains were cut in half along the midline. One half was preserved in 4% paraformaldehyde for morphology

143 analyses, whereas hippocampus and cortex were dissected from the other half and flash-frozen for western blot analyses. Brains were embedded in Tissue-Tek OCT compound and sectioned at 16 µm. Brains were then stained via Nissl staining, imaged and measurements of the hippocampus and cortex were obtained via Nikon Elements (Tokyo, Japan, RRID:

SCR_014329) software and assessed individually (Gittins and Harrison, 2004) (Cresyl Violet

Acetate, Chem-Impex International, Wood Dale, IL). For consistency, slides were numbered during sectioning, medial to lateral. Slide numbers used for each mouse were approximately the same number but bregma levels were confirmed using a brain atlas. Three serial sections per brain were assessed for each mouse at the approximate bregma level of -2 mm. The cortex was assessed using three measurements directly superior to the hippocampus. The position of the three hippocampal measurements are indicated in Figure 7K. In total, nine data points were averaged for each mouse, and each dot on the bar graphs in Figures 7E and J represents this average of one mouse. Contrast and brightness were adjusted for visualization in Figures 7A-D and F-I.

Cell Soma Size Assessment

20x magnification images of the CA1 and dentate gyrus were taken for each mouse used for the previously described morphology assessments. The same bregma level and location of regions of interest in the CA1 and the dentate gyrus was chosen for each mouse to minimize variance (Fig. 7I, red boxes). Twenty cells were measured for each mouse using

ImageJ by measuring both height and width and averaging these measurements. These averages were used as one data point for statistical analyses and visualization. Cells were randomly selected by measuring approximately every third cell counted.

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Experimental Design and Statistical Analyses

Statistical analysis was performed with GraphPad Prism 8. Tests were chosen based on the study design for each experiment. Significance is defined as a p value of 0.05 or less. Data was tested for normality with the Shapiro-Wilk test and appropriate parametric or nonparametric tests were performed. For western blot analyses, outliers were identified using the Rout method

(Motulsky and Brown, 2006). The majority of data did not have any outliers. If outliers were identified and removed, this is indicated in the figure legend. Comparisons of nest building between untreated Pten; Camkdel mice and wild type littermates were analyzed with Mann-

Whitney test. In vitro assays in neuronal cultures were analyzed by two-way ANOVA followed by

Sidak’s post hoc tests. Dose escalation studies, seizure frequency, seizure duration and total time seizing were analyzed by two-way ANOVA with repeated measures followed by Sidak’s post hoc tests, daily changes in seizure frequency, EEG power and EEG spike and spike train frequency were analyzed by mixed effect analysis followed by Sidak’s post hoc tests. Effects of drug and treatment on body weight were analyzed using three-way ANOVA with repeated measures followed by Sidak’s post hoc tests. All other assays that involved two genotypes and two different treatments were analyzed by two-way ANOVA with Tukey's post hoc tests. Sample sizes for molecular studies were determined by power analyses on pilot data using SAS ® v9.4.

Sample sizes for seizure and macrocephaly analyses were based on published sample sizes.

For all EEG, nesting and morphology studies, the treatment was blinded for the individual administering the drug and analyzing the data until analysis was completed. Each datapoint represents an independent neuronal culture or animal, respectively.

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Results

Selective inhibition of p110β rescues increased signal transduction in the context of

PTEN deficiency

In PTEN-deficient cancer cells, exaggerated PI3K signaling and tumorigenesis is mainly driven by the class 1a PI3K isoform p110β, but it is unknown if PTEN-deficient neurons have the same isoform-specificity. To answer this question, we assessed the effects of isoform-selective class 1a PI3K inhibitors on cell signaling in PTEN-deficient cultured primary neurons. We chose inhibitors that are highly specific towards one p110 isoform (IC50 at least 40-fold lower than the next lowest IC50) (Table 1), and concentrations were determined based on previous publications and our own pilot experiments ensuring that signaling in wild type neurons was minimally affected as observed with the p110β inhibitor (data not shown). We transfected primary embryonic cortical or hippocampal neurons from wild type mice with a Pten-specific small interfering ribonucleic acid (siRNA) or a scrambled control. The Pten-selective siRNA significantly reduced PTEN in hippocampal and cortical neurons (Fig. S1A-D). Three days after transfection, cells were exposed for one hour to either vehicle, a p110β-selective inhibitor

GSK2702926A (GSK6A) (Lin et al., 2012), or, in two separate experiments, either the p110δ- selective inhibitor IC-87114 (IC87) (Sadhu et al., 2003; Werzowa et al., 2011) (Fig. 1A,B) or the p110α inhibitor Alpelisib (BYL719) (Nehme et al., 2014; Wong et al., 2015) (Fig. 1C,D). Western blot analysis of phosphorylation of AKT at threonine 308, which is a downstream target of the class I PI3K pathway, was used as a readout of the pathway.

AKT phosphorylation was significantly elevated in cortical neurons after Pten siRNA transfection compared to scrambled control when treated with vehicle. Increased AKT phosphorylation was reduced to control levels with GSK6A but not with IC87 (Fig. 1A). A similar pattern was observed in primary hippocampal neurons (Fig. 1B). Likewise, the p110α-selective inhibitor BYL719 did not significantly reduce increased signaling in cortical neurons after Pten

146 siRNA transfection under conditions where GSK6A rescued elevated AKT phosphorylation (Fig.

1C). In hippocampal neurons, however, both GSK6A and BYL719 normalized elevated AKT phosphorylation after Pten siRNA transfection (Fig. 1D). Overall, these results suggest that, similar to dividing cancer cells, increased PI3K signaling is mainly mediated by the PI3K isoform p110β, and to a lesser extent by p110α in PTEN-deficient postmitotic neurons.

To assess if the selectivity for p110β is due to changes in expression levels of class 1a

PI3K isoforms in PTEN-deficient primary neurons, we next quantified protein and mRNA expression of p110α, β, and δ in cortical and hippocampal primary neurons after transfection with Pten siRNA or scrambled siRNA. Using western blot analyses, we found no significant differences in protein expression for any of the three isoforms (Fig. S1A,B,E,F). qRT-PCR specific for the three isoforms also revealed no significant difference in mRNA levels (Fig.

S1G,H).

A direct comparison of expression levels of the three isoforms, which may explain differences in drug sensitivity, is not possible with the methods used here. Apart from differential expression levels, differences in post-translational regulation by protein binding partners in

PTEN-deficient background could provide insight into the mechanisms underlying the differential responses to p110 isoform inhibitors. A recent study in cancer cell lines attributed the mechanism of how p110β drives excessive PI3K signaling when PTEN function is impaired to dysregulation of a protein complex including p110β and the adaptor protein, CRK-like (CRKL).

In cancer cells lacking PTEN, CRKL recruits p110β into this protein complex which activates

RAC signaling. RAC signaling potentially induces a positive feedback loop after activating p110β (Zhang et al., 2017). To test whether a similar mechanism occurs in neurons, we performed immunoprecipitation studies in PTEN-deficient cancer cells (PC3) and primary neurons that were treated with either scrambled or Pten-specific siRNA. We confirmed an interaction between CRKL and p110β in PC3 cells but did not detect an association in primary neurons (Fig. S2A,B). These results suggest that the mechanisms underlying p110β-selectivity

147 in the context of PTEN deficiency are different in dividing cancer cells and postmitotic neurons.

We speculate that other proteins and protein complexes affecting 110β activity, such as p85 or so far unknown protein binding partners may be altered in PTEN-deficient neurons contributing to the heightened sensitivity to p110β-selective inhibition in PTEN-deficient neurons. This hypothesis could be further tested with immunoprecipitations and mass spectrometry in the context of PTEN deficiency in the future.

Selective inhibition of p110β reduces aberrant PI3K signaling and protein synthesis in neuron-specific Pten mutant mice

After confirming that p110β-selective inhibition rescues aberrant AKT phosphorylation in

PTEN-deficient primary cortical and hippocampal neurons, we assessed cell signaling and protein synthesis in vivo in CamKIIα-Cre;Ptenfl/fl (henceforth Pten; CamKdel) mice. The CaMKIIα promoter targets forebrain excitatory neurons starting around postnatal day 14 (Tsien et al.,

1996). Pten; CamKdel mice have impairments in spatial memory and synaptic plasticity (Sperow et al., 2012), as well as spontaneous recurrent seizures reported to start at 5-6 weeks of age

(McMahon et al., 2012).

Acute sections of either somatosensory cortex or hippocampus were exposed to increasing doses of GSK6A in oxygenated artificial cerebrospinal fluid for 30 minutes. Pten;

CamKdel mice have increased AKT phosphorylation compared with littermate controls in both cortical and hippocampal slices (Fig. 2A,B). As the concentration of GSK6A increases, AKT phosphorylation decreases proportionately in Pten; CamKdel mice. Although there is a decrease in AKT phosphorylation in the littermate controls, the decrease is smaller and not proportionate to the dose of GSK6A (Fig. 2A,B). This suggests p110β is driving or partially driving aberrant PI3K signaling in PTEN-deficient cortex and hippocampus.

Next, we assessed protein synthesis, which is regulated by PI3K/mTOR signaling, in acute cortical and hippocampal slices using puromycin labeling of newly synthesized proteins

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(Gross et al., 2019; Schmidt et al., 2009). There was a significant increase in protein synthesis rates in Pten; CamKdel cortical slices compared with cortical slices from littermate controls (Fig.

2C). The addition of GSK6A to Pten; CamKdel cortical slices reduced protein synthesis to levels comparable to littermate controls (Fig. 2C). In hippocampal slices, there also was a significant increase in protein synthesis in Pten; CamKdel slices, which was significantly decreased with

GSK6A but remained increased compared to vehicle control (Fig. 2D).

We next assessed AKT phosphorylation in the slices used for protein synthesis assays.

In both hippocampal and cortical Pten; CamKdel slices, AKT phosphorylation was increased compared with littermate slices, and GSK6A reduced AKT phosphorylation to levels comparable to littermate controls (Fig. 2E,F).

P110 isoform protein expression is unchanged in Pten; CamKdel mice

Western blot analyses confirmed a decrease in PTEN in cortex and hippocampus of

Pten; CamKdel mice compared with littermate controls (Fig. S3A-D). Similar as in cultured neurons, western blot assessment of p110α, β, or δ revealed no significant differences in protein expression between Pten; CamKdel mice and controls (Fig. S3A,B,E,F).

One acute dose of GSK6A in vivo reduces aberrant AKT phosphorylation in Pten;

CamKdel mice

To test if GSK6A could be used to assess the effect of p110β-selective inhibition on seizure and behavioral phenotypes in PTEN-deficient mice, we next administered GSK6A in vivo to Pten; CamKdel mice. This dose was based on recommendations from the manufacturer and our previous results in a mouse model for FXS (Gross et al., 2019). AKT phosphorylation was increased in the cortex of Pten; CamKdel mice and one dose of GSK6A (5 mg/kg) administered intraperitoneally (i.p.) significantly reduced AKT phosphorylation to littermate control levels after one hour (Fig. 3A,B). Similarly, elevated AKT phosphorylation in the

149 hippocampus of Pten; CamKdel mice was significantly reduced to littermate control levels after one dose of GSK6A (Fig. 3C,D).

Daily administration of GSK6A extends life span in Pten; CamKdel mice

We then started dosing mice daily, as we speculated that chronic treatment may be necessary to elicit effects on seizures. Daily i.p. injections of GSK6A significantly increased survival in Pten; CamKdel mice compared with vehicle (Fig. 3E).

We noticed that daily handling and i.p. injections of the mice during chronic drug treatment led to premature death in Pten; CamKdel mice compared with mice of the same genotype which were not subjected to daily handling (data not shown). One mouse was observed having a seizure and died directly after the injection; however, we are still investigating additional causes to this mortality. In order to successfully deliver GSK6A to Pten;

CamKdel mice over long administration periods without increasing mortality, we next took advantage of the oral availability of GSK6A (Gross et al., 2019) by providing the drug in small, single-serve portions of peanut butter. All mice, regardless of genotype, were highly receptive to this dosing technique. No increased lethality was observed with this dosing strategy. Based on recommendations from the manufacturer, two different doses were initially tested (2.5 mg/kg and 5 mg/kg). The higher dose was chosen due to increased efficacy to reduce AKT phosphorylation to control levels in these pilot studies and based on recommendations by

GlaxoSmithKline (Fig. S4).

Daily oral administration of GSK6A improves nest building behavior and reduces aberrant PI3K signaling in Pten; CamKdel mice

We next assessed how chronic oral treatment with GSK6A affects nest building behavior in Pten; CamKdel mice. Nest building has been shown to be altered in mouse models of neurodevelopmental disorders (Gross et al., 2015), but to our knowledge, has not been

150 assessed in mice with neuronal PTEN deficiency yet. Here, we show that Pten; CamKdel mice shred on average less nest building material compared with littermate controls (Fig. 4A). One week of daily oral administration of GSK6A significantly increases nest building behavior in

Pten; CamKdel mice compared with vehicle-treated Pten; CamKdel mice without affecting littermate controls (Fig. 4B). Note that some of the mice had EEG electrodes implanted during nesting.

After completion of nest building assays, mice were sacrificed and cortex and hippocampus were used for western blot analyses to quantify PI3K/mTOR signaling (Fig. 4C-J,

S5). We first assessed phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1) as one of the most direct downstream targets of PI3K. We detected significantly increased

PDK1 phosphorylation in Pten; CamKdel cortex, which was reduced to littermate control levels after one week of GSK6A administration (Fig. 4E). Interestingly, the effect was less pronounced in the hippocampus, showing a significant effect of genotype on PDK1 phosphorylation and an interaction between genotype and treatment, but no significant differences in pairwise comparisons (Fig. 4H). To distinguish between effects on signaling activity of mTORC1 and mTORC2, we assessed phosphorylation of AKT at threonine 308 (mTORC1) and serine 473

(mTORC2). Both were significantly increased in Pten; CamKdel cortex and hippocampus compared with littermate controls and normalized to vehicle-treated control levels after GSK6A administration, except for pAKT (T308), which was still significantly higher in Pten; CamKdel after GSK6A treatment compared with littermate controls (Fig. 4F,G,I,J). The effect of the drug was more pronounced in cortex (4F,G) compared to hippocampus (4I,J). To further analyze mTORC1 downstream signaling, we quantified phosphorylation of p70 S6 Kinase (S6K) and S6

Ribosomal Protein (S6). We detected significant effects of genotype on phosphorylation of S6K in cortex and hippocampus, with on average increased S6K phosphorylation in PTEN-deficient mice (Fig. S5A-D). We also detected a significant effect of treatment in hippocampus (Fig. S5D, reduction of S6K phosphorylation) but not in cortex (Fig. S5C), and no differences in pairwise

151 comparisons in either tissue. In contrast to previous reports (Chen et al., 2019), we did not detect consistent differences in S6 phosphorylation in either cortex or hippocampus (Fig.

S5A,B,E,F). S6 is regulated by many different signaling pathways and can be altered through handling or environment (Biever et al., 2015), which may underlie the lack of baseline differences or drug effects in this study. Of note, Chen et al. (2019) showed that normalization of

S6 phosphorylation may not be needed to reduce seizure frequency, justifying our approach to test the effect of p110β inhibition on seizure frequency.

GSK6A reduces seizure frequency in Pten; CamKdel mice

We next assessed how chronic treatment with GSK6A affects seizure activity in Pten;

CamKdel mice. A previous study has reported that Pten; CamKdel develop spontaneous recurrent seizures between 5 and 6 weeks of age (McMahon et al., 2012). We first confirmed the seizure phenotype in Pten; CamKdel mice by 24/7 cortical electroencephalography (EEG) and video recording. In our hands, Pten; CamKdel mice started to exhibit class 4-5 tonic-clonic seizures at approximately 8 weeks, but the timing was highly variable (data not shown). To test the effect of GSK6A on seizure frequency, we implanted Pten; CamKdel and littermate controls at 7-8 weeks of age with cortical surface electrodes using a wireless transmitter system. After occurrence of seizures was confirmed by 24/7 video-EEG recording, seizures were recorded for

5 consecutive days to assess baseline seizure frequency. Starting at day 6 after detection of the first seizure, mice were administered once daily with either GSK6A or vehicle for 7 days (in peanut butter, timeline shown in Fig. 5A, example seizure shown in Fig. 5B). A total of 13 control litter mates were subjected to cortical surface electrode implants. No seizures were detected in any of these animals (Table 2).

During the 5 days of assessment prior to GSK6A administration, there was no significant difference in the frequency of seizures observed between GSK6A- or vehicle-treatment groups.

By contrast, there were significantly fewer seizures in GSK6A-treated mice compared with

152 vehicle-treated mice during the final 5 days of dosing (Fig. 5C). Daily assessment of average seizure frequency showed that the two treatment groups were equal prior to treatment and began to separate after daily treatment started (Fig. 5D, box).

In order to evaluate if the differences seen in response to treatment were due to model variability, we assessed the average seizure frequency for each individual mouse prior to and during the dose administration period. All mice that received vehicle showed an increase in seizures during the treatment period; however, 7 of 8 GSK6A-treated mice showed a decrease in seizure frequency (Fig. 5E). Post hoc, seizure quantity was assessed for the first 5 days of the study and mice were grouped by sex. There was no significant difference in the average frequency of seizures between females and males (Fig. 5F).

Seizure duration was assessed and compared for each treatment group before and after treatment started. There was no significant difference in the average duration of seizures between the two treatment groups before and after treatment (Fig. 5G). The total time spent seizing during the 5 days of assessment was also not significantly different (Fig. 5H).

To further assess the effects of GSK6A on brain activity in Pten; CamKdel mice we analyzed

EEG power, spikes and spike trains during interictal phases. The GSK6A-treated mice had overall lower gamma EEG power, and reduced spike and spike train frequency during the last days of treatment compared with vehicle-treated mice (Fig. S6). Due to confounding factors such as cardiac signals (heartbeat) or EEG readings with excessive artifacts and excessive seizures, many mice had to be excluded from this analysis. Although the data supported the excitability-suppressing effect of GSK6A, the low sample number (3 per group) combined with the inherent variability of EEG signal made data interpretation difficult. EEG power, spike, and spike train analysis performed by Dr. Durgesh Tiwari, PhD.

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Chronic treatment with GSK6A reduces increased AKT phosphorylation and brain weight in Pten; Gfapdel mice

In humans, PTEN mutations have been associated with macrocephaly (Frazier et al.,

2015; McBride et al., 2010). The Pten; CamKdel mouse model was originally reported to not have a macrocephaly phenotype (Sperow et al., 2012); therefore, we used a different Pten mouse model for this assessment, Gfap-Cre; Ptenfl/fl (henceforth, Pten; Gfapdel), which has a macrocephaly and neuronal hypertrophy phenotype (Backman et al., 2001; Kwon et al., 2001).

Pten; Gfapdel mice and their littermate controls were treated with either GSK6A or vehicle, administered intraperitoneally, for four weeks, beginning at P28. To confirm the effectiveness of the drug to reduce signaling and exclude compensation effects after chronic treatment, we performed western blot analyses of cortical and hippocampal lysates of some of these mice.

These analyses showed that Pten; Gfapdel mice have a similar decrease in PTEN and increase in AKT phosphorylation as seen in Pten; CamKdel mice, which was reduced to or below control levels after daily dosing with GSK6A for four weeks (Fig. 6A-F).

Next, we assessed the brain weight to body weight ratios for Pten; Gfapdel mice as an initial assessment of macrocephaly. The brain-to-body ratio was significantly increased in Pten;

Gfapdel mice treated with vehicle compared with littermate controls, which was normalized to littermate control levels with GSK6A (Fig. 6G). Importantly, overall body weight was not affected by genotype or treatment, and all mice gained weight over the 4-week treatment period (Fig.

6H).

GSK6A reduces cortical dysplasia and hippocampal neuronal hypertrophy in Pten;

Gfapdel mice

We next analyzed the effects of chronic GSK6A treatment on abnormal morphology of the cortex and hippocampus in Pten; Gfapdel mice. The thickness of the cortical plate of Pten;

Gfapdel mice was significantly increased after 4 weeks of vehicle administration compared with

154 littermate controls and reduced to control levels after 4 weeks of GSK6A treatment (Fig. 7A-E).

Similar as the cortical plate, the hippocampi of Pten; Gfapdel mice were significantly enlarged in

Pten; Gfapdel that received vehicle for four weeks compared with littermate controls. Although there was an overall decrease in hippocampal size after GSK6A treatment in Pten; Gfapdel mice, hippocampi of Pten; Gfapdel mice were still significantly larger than those of vehicle- treated controls (Fig. 7F-K). To test if GSK6A reduced neuronal hypertrophy in the hippocampus, we assessed average soma size of dentate gyrus granule cells and of CA1 pyramidal cells. The average soma size of granule cells of the dentate gyrus, which was significantly enlarged in Pten; Gfapdel mice compared with littermate controls as reported previously (Kwon et al., 2001), was reduced to littermate control levels after GSK6A treatment

(Fig. 7L). There were also overall effects of genotype and drug treatment on soma size of CA1 pyramidal neurons, indicating that soma size is increased in the CA1 of PTEN-deficient mice and reduced with GSK6A treatment, but no significant differences in a pairwise comparisons were detected (Fig. 7M).

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Discussion

In this work, we have demonstrated that inhibition of one of the four class 1 PI3K catalytic isoforms expressed in the brain, p110β, reduces molecular defects and seizures, and normalizes nesting behavior, cortical plate thickness and hippocampal neuronal hypertrophy in mice with neuron-specific PTEN deficiency. Our results create a compelling argument for investigation of p110β inhibition as potential treatment strategy in humans with PTEN mutations accompanied by macrocephaly and seizures. Together with our previous studies in FXS, they also provide strong evidence that p110β may be a key player and shared treatment target in different types of monogenic brain disorders.

PI3K is the enzymatic counteractor of PTEN’s lipid phosphatase activity. Inhibition of

PI3K is thus expected to correct the core molecular defect caused by loss of PTEN in neurons; however, PI3K activity also plays roles in other processes that are essential for normal body function, such as cell proliferation, survival and migration. Here, we took advantage of the fact that there are three closely related class 1a isoforms expressed throughout the body including the brain, which have unique and overlapping functions (Vanhaesebroeck et al., 2010). A previous study has shown that these isoforms can compensate for loss or reduction of one isoform for essential cellular functions such as proliferation and survival (Foukas et al., 2010), suggesting that a single isoform-targeted strategy may not have severe side effects. In line with this assumption, we did not detect any detrimental effects on overall health in the mice as assessed by daily health monitoring and tri-weekly body weight recordings. Of note, chronic inhibition of the downstream target mTOR inhibitor, which is currently evaluated as treatment strategy for individuals with PTEN mutations in clinical trials, has been shown to significantly decrease body weight in PTEN-deficient mice (Sunnen et al., 2011; Zhou et al., 2009), whereas four weeks of daily GSK6A treatment did not affect body weight in mice. Longer term studies

156 are needed to further evaluate the effect of chronic p110β inhibition on body weight and overall health, and whether it may have fewer side effects than mTOR inhibition.

Our results provide evidence that selectively inhibiting one specific PI3K isoform in

PTEN-deficient brain disorders while leaving the others intact improves PTEN-associated neuronal phenotypes in mice. This suggests that the other isoforms, p110α, γ and δ, do not compensate for reduced p110β in the context of neuronal function, and that p110β may have a unique role in mediating dysregulated neuronal PI3K/AKT signaling defects in PTEN-associated neurodevelopmental brain disorders. This hypothesis is further supported by our in vitro studies indicating that, similar to dividing tumor cells, molecular defects associated with PTEN- deficiency in postmitotic neurons are largely mediated by p110β but not the other class 1a PI3K isoforms (Jia et al., 2008). Interestingly, p110α inhibition with BYL790 also reduced increased

AKT phosphorylation in PTEN-deficient hippocampal neurons (but not in cortical neurons), suggesting that, similar to cancer, depending on the cell type and environment, p110α can mediate PTEN-associated defects (Berenjeno et al., 2012). In line with this assumption, our biochemical and brain morphological analyses show slight differences in the effect of GSK6A on cortical versus hippocampal signal transduction and dysplasia. Although phenotypes were reduced in both brain regions with the drug, the effects in the cortex were often more pronounced than in the hippocampus, suggesting that apart from p110β an additional PI3K isoform, most likely p110α, plays a role in altered signaling in PTEN-deficient hippocampal neurons. It is unclear whether the p110α inhibitor BYL790 crosses the blood brain barrier and whether the efficiency to cross it is comparable to GSK6A’s. To our knowledge, no other brain- permeable p110α inhibitor has been reported. We were thus not able to compare the efficacies of these two drugs in vivo. Concentrations used for in vitro studies were chosen based on previous publications and our pilot experiments. Limitations of this study are that we did not test higher concentrations of the other isoform-selective inhibitors in vitro and that we were not able to assess the effect of p110α inhibition in vivo. While our results support p110β as a potential

157 treatment for PTEN-associated epilepsy, we cannot rule out that p110α or p110δ inhibition would be beneficial as well. Additionally, given the feedback loops which also regulate the PI3K pathway, there may be utility in inhibition of additional pathways in conjunction with PI3K inhibition. Future studies are needed to further evaluate these possibilities.

We showed that p110β inhibition over several days reduced the occurrence of spontaneous seizures in Pten; CamKdel mice. The treatment effects were consistent overall (all vehicle-treated mice had increased seizure frequency, whereas 7 out of 8 GSK6A-treated mice had reduced seizure frequency after treatment), but there was high variability in this model. For example, the age of seizure onset differed between mice. Although mice were implanted at the same age, in some mice, we detected seizures at the day after implantation, whereas in others it took up to 18 days until a first seizure was detected. While all mice had spontaneous seizures for at least five days until treatment started, we cannot exclude that the variability in time seizing before treatment influenced the effectiveness of the drug. Moreover, the mortality rate for this procedure was high (Table 2), and the quantity of seizures an individual mouse experienced was also highly variable. Although the study was blinded, these factors could potentially skew the results. Seizures were reduced but not eliminated with GSK6A, and future studies are needed to evaluate if prolonged treatment or a combinatorial approach targeting two different

PI3K isoforms may further reduce or even stop seizures. A previous study has shown that inhibition of all class I PI3K isoforms (p110α,β,γ,δ) using a pan PI3K inhibitor suppresses seizures in epilepsy caused by mutations in the gene coding for p110α, PIK3CA (Roy et al.,

2015). While broad inhibition of all PI3K activity is most likely not feasible as a clinical therapy because of detrimental side effects, this research, together with our results that a p110α- selective inhibitor reduces AKT phosphorylation in PTEN-deficient hippocampal neurons in vitro, suggests that p110α activity may contribute to the seizure phenotype and that a combination of p110β and p110α inhibitors could lead to a more comprehensive reduction of seizures. This

158 strategy should be tested in the future, when brain-permeable p110α inhibitors become available.

Our experiments do not show whether p110β inhibition is merely anti-convulsant or has a disease-modifying effect. To address this question, additional assessments of seizure frequency after GSK6A treatment has ended are required. Due to the high mortality rate of the model, however, we were not able to assess longer time periods. The present study was designed to mimic a treatment in humans, which would start after the occurrence of seizure symptoms. Future studies could evaluate if an earlier, pre-symptomatic start of treatment has disease-modifying effects in these mice. Alternatively, a potential disease-modifying effect after treatment has ended could be tested in another, less severe mouse model of reduced neuronal

PTEN expression.

A four-week treatment with GSK6A starting at P28 reduced cortical plate thickness, dentate granule cell and CA1 pyramidal cell soma size, as well as the brain to body weight ratio in a mouse model for PTEN-associated focal dysplasia to control levels; by contrast, GSK6A only partially reduced hippocampal size, which was still significantly larger than in vehicle- treated controls. The fact that p110β inhibition did normalize dentate granule cell soma size and had a moderate effect on CA1 pyramidal soma size and overall hippocampal size suggests that extended treatment may be required for a comprehensive rescue of hippocampal morphology.

Alternatively, a combinatorial treatment approach using p110β and p110α inhibitors, as discussed above in the context of seizures, could be used. Of note, although the PTEN deletion in neurons of the dentate gyrus and the CA1 is extensive, not every cell is a Pten knockout cell.

The detected effects might thus have been diluted with a small percentage of wild type cells.

Several mouse models of neuronal PTEN deficiency have been subjected to mTOR inhibition with rescue of many phenotypes, including seizures and macrocephaly (Chen et al.,

2019; Getz et al., 2016; Kwon et al., 2003; Ljungberg et al., 2009; Sunnen et al., 2011; Zhou et

159 al., 2009). This work argues in favor of mTOR inhibition for individuals with brain disorders caused by PTEN mutations; however, mTOR is several steps downstream of PTEN. Notably, both inhibition of mTORC1 or mTORC2 has been shown to be effective in mouse models (Chen et al., 2019; Nguyen et al., 2015). The approach of the present study is expected to restore deficits further upstream, reduces mTORC1- and mTORC2-mediated signaling, and may thus be used as an alternative or add-on strategy. It has been shown in PTEN-deficient neuronal cultures that AKT inhibition is more efficient at rescuing hypertrophy than mTOR inhibition, arguing in favor of inhibition upstream of mTOR (Nikolaeva et al., 2017). Future studies in mouse models are needed to directly compare the efficacy of both treatment strategies and to compare the potential negative side effects.

Our study may have impact beyond PTEN-associated epilepsy because it supports an important role of p110 isoforms in brain function and development. The PI3K/mTOR pathway is a hotspot of mutations and alterations in several neurodevelopmental disorders, including autism and epilepsy, and in most cases the molecular defect or mutation is upstream of mTOR

(Crino, 2016; Gross and Bassell, 2014; Iffland et al., 2019; Poopal et al., 2016; Rademacher and Eickholt, 2019; Schick et al., 2006; Schick et al., 2007). Class I PI3K isoforms signal upstream of mTOR, play unique isoform-specific roles in the body, in particular in the brain and signal downstream of certain membrane receptors (Gross and Bassell, 2014; Vanhaesebroeck et al., 2010). These functions also tie p110 isoforms to specific neuronal disorders: p110α is associated with Alzheimer’s disease (Bosco et al., 2011), p110β is a molecular defect and promising treatment target in FXS (Gross et al., 2019) and possibly other forms of autism

(Cusco et al., 2009), and p110δ is altered in schizophrenia (Law et al., 2012). Our work further advocates for the investigation of potential usage of PI3K isoform-specific inhibition to treat disease mechanisms in neurological disorders.

160

Acknowledgements:

This research was supported by NIH grants 1R21HD093033 (CG), 1R01NS092705

(CG), and 2R01NS065020 (SCD), NIH NCATS Award UL1 TR001425, and a NARSAD

Independent Investigator Award from the Brain and Behavior Research Foundation (CG). The authors would like to thank Andrea Shugar, Andrew Snider, Jeffrey Rymer, and Lindsay Carter for their technical assistance, Dr. Paul Horn for statistical power analyses performed for molecular studies, and Dr. Gary Bassell for helpful comments on an earlier version of this manuscript. The authors would like to acknowledge Jonah White for the idea of using peanut butter for delivery of oral compounds in an environment which does not evoke stress. We would also like to thank Dr. Matthew Weston for the donation of Pten; Gfapdel mice and Dr. Ralph

Rivero for the contribution of GSK6A. GSK6A was obtained through a Material Transfer

Agreement from GlaxoSmithKline. All studies were conducted in accordance with the GSK

Policy on the Care, Welfare and Treatment of Laboratory Animals and were approved by the

Institutional Animal Care and Use Committee at CCHMC. GSK6A is covered by US patents

20130157977A1 and 8778937B2.

161

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Figures and tables:

173

Figure 1: Elevated AKT phosphorylation in PTEN-deficient neuronal cultures is reduced to control levels with selective p110β inhibition. (A, B) Pten siRNA significantly increases

AKT phosphorylation at threonine 308 three days after transfection in both primary cortical (A) and hippocampal neurons (B). The p110β inhibitor GSK6A (1μM) reduces AKT phosphorylation to levels comparable to those of control (scramble siRNA-transfected) neurons, whereas the p110δ inhibitor IC87 (2μM) does not reduce AKT phosphorylation (two-way ANOVA with Sidak’s post hoc tests: A, p(interaction)=0.046, F(2,54)=3.26; p(treatment)=0.027, F(2,54)=3.87; p(siRNA)=0.001, F(1,54)=11.98; **p=0.007, *p=0.017, p(GSK)>0.999; n=10; B, p(interaction)=0.084, F(2,52)=2.60; p(treatment)=0.071, F(2,52)=2.78; p(siRNA)<0.0001,

F(1,52)=17.47; **p=0.002, *p=0.011, p(GSK)=0.921; n(scr GSK6A)=9, all others n=10). (C, D) In contrast to GSK6A, the p110α inhibitor BYL719 (0.05µM) does not reduce AKT phosphorylation in primary cortical neurons (C). Both GSK6A (1μM) and BYL719 (0.05μM) reduce AKT phosphorylation in primary hippocampal neurons (D) (two-way ANOVA with Sidak’s post hoc tests: C, p(interaction)=0.349, F(2,60)=1.07; p(treatment)=0.001, F(2,60)=7.84; p(siRNA)<0.001, F(1,60)=16.66; *p(vehicle)=0.012, *p(BYL719)=0.016, p(GSK6A)=0.577; n=11;

D, p(interaction)=0.476, F(2,60)=0.75; p(treatment)=0.269, F(2,60)=1.34; p(siRNA)=0.001,

F(1,60)=11.67; *p(vehicle)=0.014, p(GSK)=0.498, p(BYL)=0.262; n=11). One statistical outlier was removed from B (scr GSK6A); ns: not significant. N are independent neuronal cultures.

Error bars represent SEM. Example western blots are shown at the top in each panel.

174

175

GSK6A on AKT phosphorylation in acute cortical (A) and hippocampal (B) slices from Pten;

CamKdel mice and their littermate controls illustrates that the p110β inhibitor GSK6A reduces

AKT phosphorylation in a dose-dependent manner in Pten; CamKdel mice. This dose- dependent decrease is less pronounced in control littermates (repeated measures two-way

ANOVA with Sidak’s post hoc tests: A, p(interaction)=0.022, F(5,60)=2.85; p(treatment)<0.0001,

F(5,60)=8.37; p(genotype)=0.894, F(1,12)=0.02; p(subject)=0.605, F(12,60)=0.85; *p=0.025; n=8 (Pten; CamKdel) and 6 (control); B, p(interaction)=0.008, F(5,50)=3.56; p(treatment)<0.001,

F(5,50)=6.83; p(genotype)=0.149, F(1,10)=2.49; p(subject)=0.998, F(10,50)=0.16; *p=0.030; n=7 (Pten; CamKdel) and 5 (control)). (C,D) 1μM GSK6A significantly reduces elevated protein synthesis rates in cortical (C) and hippocampal (D) slices from Pten; CamKdel mice after 30 minutes of exposure (two-way ANOVA with Tukey’s post hoc tests: C, p(interaction)=0.188,

F(1,26)=1.83; p(treatment)=0.066, F(1,26)=3.68; p(genotype)=0.001, F(1, 26)=13.62; **p=0.007; n=8 (Pten; CamKdel) and 7(control); D, p(interaction)<0.001, F(1, 23)=14.82; p(treatment)=0.031, F(1,23)=5.28; p(genotype)<0.0001, F(1,23)=44.3; ****p<0.0001, **p=0.002;

*p=0.022; n(Pten; CamKdel vehicle)=6, all others n=7). Puromycin-specific signal was normalized to AKT. V=vehicle, G=GSK6A, No=no Puromycin. (E,F) 1μM GSK6A reduces elevated AKT phosphorylation in cortical (E) and hippocampal (F) slices from Pten; CamKdel mice after 70 minutes of exposure (two-way ANOVA with Tukey’s post hoc tests: E, p(interaction)=0.064, F(1,26)=3.75; p(treatment)=0.095, F(1,26)=3.00; p(genotype)=0.002,

F(1,26)=11.88; **p=0.004; n=8 (Pten; CamKdel) and 7 (control); F, p(interaction)=0.022,

F(1,20)=6.21; p(drug)=0.014, F(1,20)=7.27; p(genotype)=0.027, F(1,20)=5.67; *p=0.013,

**p=0.008; n=6). One statistical outlier was removed from D (vehicle Pten; CamKdel). N denotes

176 individual animals. Error bars represent SEM. Representative western blots shown at top in each panel.

177

Figure 3: Administration of GSK6A reduces elevated AKT phosphorylation and increases survival in Pten; CamKdel mice. (A-D) Intraperitoneal injection of 5mg/kg GSK6A reduces

178 elevated AKT phosphorylation in Pten; CamKdel mice to levels comparable to littermate controls in both cortical (A,B) and hippocampal lysates (C,D) one hour after dose administration (two- way ANOVA with Tukey’s post hoc test: B, p(interaction)=0.020, F(1,8)=8.35; p(treatment)=0.045, F(1,8)=5.62; p(genotype)=0.009, F(1,8)=11.63; *p=0.024; **p=0.009; D, p(interaction)=0.033, F(1,8)=6.61; p(treatment)=0.069, F(1,8)=4.43; p(genotype)=0.036,

F(1,8)=6.36; *p(Pten; CamKdel vehicle - control vehicle)=0.029; *p(Pten; CamKdel vehicle -

Pten; CamKdel GSK6A)=0.043). Representative western blots are shown in A and C. (E) Daily intraperitoneal injections of GSK6A significantly increase survival of Pten; CamKdel mice compared to daily injections of vehicle (Kaplan-Meier survival curve comparison, Mantel-Cox test: **p=0.005; n=4 (vehicle) and 5 (GSK6A)). Error bars represent SEM.

179

Figure 4: GSK6A improves nest building behavior and reduces aberrant PI3K signaling in

Pten; CamKdel mice. (A) Pten; CamKdel mice shred significantly less nesting material than their littermate controls during a 2-hour period (Mann-Whitney test, **p=0.002, n(Pten;

CamKdel)=16, n(control)=8). (B) Daily oral administration of GSK6A in peanut butter for one week significantly improves nesting behavior in Pten; CamKdel mice compared with vehicle- treated Pten; CamKdel mice but not in littermate controls (two-way ANOVA with Tukey’s post hoc tests: p(interaction)=0.030, F(1,42)=5.05; p(treatment)=0.017, F(1,42)=6.2; p(genotype)=0.471, F(1,42)=0.53; **p=0.004; n=14 (Pten; CamKdel-Vehicle), 13 (Pten;

CamKdel-GSK6A), 9 (Control-Vehicle), 10 (Control-GSK6A)). (C,D) Representative western

180 blots from the cortex (C) and hippocampus (D) collected from mice after one week of daily oral

GSK6A or vehicle administration. (E) GSK6A reduces elevated PDK1 phosphorylation in Pten;

CamKdel cortex to littermate control levels (two-way ANOVA with Tukey’s post hoc tests: p(interaction)=0.173, F(1,24)=1.98; p(treatment)=0.038, F(1,24)=4.84; p(genotype)=0.003,

F(1,24)=11.03; *p=0.014; n=7). (F) One week of GSK6A treatment reduces elevated AKT phosphorylation at threonine 308 in Pten; CamKdel cortex, but phosphorylation levels are still increased compared with vehicle control (two-way ANOVA with Tukey’s post hoc tests: p(interaction)=0.091, F(1,24)=3.11; p(treatment)=0.073, F(1,24)=3.53; p(genotype)<0.0001,

F(1,24)=34.01; ****p<0.0001, *p=0.046; n=7). (G) Daily administration of GSK6A for one week significantly reduces elevated AKT phosphorylation at serine 473 in Pten; CamKdel cortex to littermate control levels (two-way ANOVA with Tukey’s post hoc tests: p(interaction)=0.024,

F(1,23)=5.84; p(treatment)=0.068, F(1,23)=3.66; p(genotype)=0.010, F(1,23)=7.89; **p=0.007;

*p=0.030; n(Pten; CamKdel vehicle)=6, n=7 all other groups). (H) PDK1 phosphorylation is increased in Pten; CamKdel hippocampus compared to littermate controls, which is not corrected by GSK6A (two-way ANOVA with Tukey’s post hoc tests: p(interaction)=0.737,

F(1,24)=0.12; p(treatment)=0.134, F(1,24)=2.40; p(genotype)=0.003, F(1,24)=10.99; n=7). (I)

GSK6A reduces elevated AKT phosphorylation at threonine 308 in Pten; CamKdel hippocampus to littermate control levels (two-way ANOVA with Tukey’s post hoc tests: p(interaction)=0.044, F(1,24)=4.52; p(treatment)=0.214, F(1,24)=1.63; p(genotype)=0.002,

F(1,24)=12.40; **p=0.0028; n=7). (J) GSK6A reduces elevated AKT phosphorylation at serine

473 in Pten; CamKdel hippocampus to littermate control levels (two-way ANOVA with Tukey’s post hoc tests: p(interaction)=0.232, F(1,24)=1.50; p(treatment)=0.065, F(1,24)=3.74; p(genotype)<0.0001, F(1,24)=22.92; **p=0.002; n=7). One statistical outlier was removed in G

(Pten; CamKdel vehicle). Error bars represent SEM.

181

Figure 5: GSK6A administration reduces seizure frequency in Pten; CamKdel mice. (A)

Timeline depicting experimental design for GSK6A treatment of Pten; CamKdel mice and

182 subsequent seizure assessment. (B) A representative EEG trace showing a spontaneous seizure in a Pten; CamKdel mouse. (C) There is no significant difference in average number of seizures per day for 5 days prior to treatment in the two treatment groups. During the 7 days of treatment, GSK6A significantly reduces average seizure frequency per day compared to vehicle

(repeated measures two-way ANOVA with Sidak’s post hoc tests: p(interaction)=0.023,

F(1,14)=6.52; p(time)=0.351, F(1,14)=0.93; p(treatment)=0.141, F(1,14)=2.44; p(subject)=0.010,

F(1,14)=3.66, *p=0.032; n=8). (D) No difference in seizure frequency at baseline (days 1-5) is detected when comparing the two treatment groups. During treatment, seizure frequency is reduced in GSK6A-treated mice compared to vehicle-treated mice with a significant difference in seizure frequency between groups on the final day of drug administration (day 12) (mixed- effects analysis with Sidak’s post hoc tests: p(time)=0.195, F(11,170)=1.36; p(treatment)=0.087,

F(1,16)=3.32; p(time x treatment)=0.036, F(11, 170)=1.95; **p=0.002; vehicle: n=9 for days 1-

11, n=8 for day 12; GSK6A: n=9 for days 1-9, n=8 for days 10-11, n=7 for day 12). Treatment days are depicted by a box. Note that mice that were excluded from analysis in C due to premature death at days 10-12 are included in mixed-effects analysis in D. (E) Individual mouse analysis of average seizure frequency per day before and during treatment. (F) Seizure frequency was similar in untreated female and male mice (unpaired t-test: p=0.592, t(18)=0.55; n=9 female; n=11 male). (G) The average duration of seizures per animal was not significantly different between genotype and treatment groups (repeated measures two-way ANOVA with

Sidak’s post hoc tests: p(interaction)=0.969, F(1,14)=0.002; p(time)=0.460, F(1, 14)=0.58; p(treatment)=0.423, F(1, 14)=0.68; n=8). (H) Total time of seizure activity during the 5 days of assessment was not significantly different between genotype and treatment groups (repeated measures two-way ANOVA with Sidak’s post hoc tests: p(interaction)=0.754, F(1,14)=0.10; p(time)=0.283, F(1, 14)=1.25; p(treatment)=0.582, F(1, 28)=0.32; n=8). Error bars in D represent SEM. Box-and-whisker plots show minimum to maximum (whiskers), 25th to 75th percentile (boxes) and median (line).

183

Figure 6: Chronic GSK6A administration reduces enhanced AKT phosphorylation and normalizes brain weight in Pten; Gfapdel mice. (A-F) PTEN expression is reduced (C,D) and

AKT phosphorylation at threonine 308 increased (E,F) in cortex (A,C,E) and hippocampus

(B,D,F) of Pten; Gfapdel mice. Four weeks of daily i.p. injections with 5mg/kg GSK6A significantly reduces increased AKT phosphorylation in cortex and hippocampus in Pten;

Gfapdel mice (E,F). Representative western blots shown in A (cortex) and B (hippocampus);

V=Vehicle, G=GSK6A (C,D unpaired t-tests: C, **p=0.004, t(4)=6.13; D, **p=0.003, t(4)=6.63;

184 n=3; E,F two-way ANOVA with Tukey’s post hoc tests: E, p(interaction)=0.052, F(1,8)=5.23; p(treatment)=0.006, F(1,8)=13.97; p(genotype)=0.068, F(1,8)=4.44; *p=0.012; n=3; F, p(interaction)=0.188, F(1,8)=2.07; p(treatment)<0.0001, F(1,8)=120.9; p(genotype)=0.0008,

F(1,8)=27.75; **p=0.006, ***p=0.0001; *p=0.016, n=3). (G) Brain to body weight ratios

(expressed in percent) are significantly increased in Pten; Gfapdel mice compared with littermate controls. Chronic GSK6A treatment for 4 weeks reduces ratios to levels comparable to littermate controls (two-way ANOVA with Tukey’s post hoc tests: p(interaction)=0.017,

F(1,26)=3.82; p(treatment)=0.254, F(1,26)=1.36; p(genotype)=0.044, F(1,26)=1.36; *p=0.014; n=9 (Pten; Gfapdel) and 6 (control)). (H) Body weight was not significantly altered by genotype or treatment, and all mice gained weight over the 4-week treatment period (repeated measures three-way ANOVA with Sidak’s post hoc tests: p(time)<0.0001, F(1,29)=99.32; p(genotype)=0.343, F(1,29)=0.93; p(treatment)=0.328, F(1,29)=0.99; p(time x genotype)=0.480,

F(1,29)=0.51; p(time x treatment)=0.428, F(1,29)=0.65; p(genotype x treatment)=0.395,

F(1,29)=0.75; p(time x genotype x treatment)=0.305, F(1,29)=1.09; all pairwise comparisons before-after p≤0.003; all pairwise comparisons veh-GSK6A or ctr-PTEN p>0.83, n=7 (Veh Ctr),

6 (GSK6A Ctr), 10 (Veh Pten), 10 (GSK6A Pten)). Error bars represent SEM.

185

186

Figure 7: Intraperitoneal GSK6A administration reduces cortical enlargement and hippocampal soma size to control levels in Pten; Gfapdel mice. (A-E) The cortices of vehicle-treated Pten; Gfapdel mice are enlarged compared with littermate controls. GSK6A administered at 5mg/kg daily (i.p.) for four weeks reduces cortical enlargement to control levels

(E, two-way ANOVA with Tukey’s post hoc tests: p(interaction)=0.062, F(1,26)=3.81; p(drug)=0.254, F(1,26)=1.36; p(genotype)=0.054, F(1,26)=4.08; *p=0.043; n=9 (Pten; Gfapdel), n=6 (control)). Assessments consisted of three measurements directly superior to the hippocampus at bregma level -2 mm in three serial sections per mouse. One data point represents the average of these nine measurements. Example images are shown in A-D, scale bar is 200 μm. (F-K) Hippocampal size is significantly increased in vehicle-treated Pten; Gfapdel mice compared with vehicle-treated control littermates. Chronic GSK6A treatment for 4 weeks shows a trend towards reducing hippocampal size; however, GSK6A-treated Pten; Gfapdel mice still have significantly larger hippocampi than vehicle-treated controls (J, two-way ANOVA with Tukey’s post hoc tests: p(interaction)=0.016, F(1,25)=6.71; p(drug)=0.518, F(1,25)=0.43; p(genotype)<0.0001, F(1,25)=37.26; ****p<0.0001, **p=0.004; n=9, Pten; Gfapdel; 8, control).

The location of measurements is represented by red lines in (K). Quantifications show averages of all three measurements in three consecutive sections, yielding nine measurements per data point. Example images are shown in F-I; scale bar is 200 μm. (L) Enlarged soma size of granule cells in the dentate gyrus of Pten; Gfapdel mice is reduced to vehicle control levels after chronic

GSK6A treatment (two-way ANOVA with Tukey’s post hoc tests: p(interaction)=0.865,

F(1,22)=0.03; p(drug)=0.109, F(1,22)=2.78; p(genotype)=0.0001, F(1,22)=21.26; **p=0.0095; n=6 (Veh Ctr), 7 (GSK Ctr), 8 (Veh, Pten), 5 (GSK, Pten)). (M) Soma size of pyramidal cells in the CA1 region is significantly increased in Pten; Gfapdel mice compared with littermate controls and significantly reduced in both groups after chronic GSK6A treatment. No significant differences in pairwise comparisons were detected (two-way ANOVA with Tukey’s post hoc tests: p(interaction)=0.521, F(1,22)=0.43; p(drug)=0.042, F(1,22)=4.65; p(genotype)=0.019,

187

F(1,22)=6.37; n=6 (Veh Ctr), 7 (GSK Ctr), 8 (Veh Pten), 5 (GSK Pten)).The location of soma size measurements for L and M is depicted by the red boxes in I. Error bars represent SEM.

188

Figure S1: mRNA and protein expression of p110 isoforms remain unchanged in primary neuronal cultures after Pten siRNA transfection. (A,B) Example western blots of cortical (A) and hippocampal (B) primary neurons show reduced PTEN expression and no difference in p110α, β, or δ protein levels after Pten siRNA transfection. (C,D) PTEN is significantly reduced after Pten siRNA transfection in cortical neurons (C) and hippocampal neurons (D) (unpaired t- tests: C, ***p<0.0001, t(20)=7.27; n=11; D, ***p<0.0001, t(18)=18.01; n=10). (E, F) PTEN levels do not affect p110 isoforms in cortical neurons (E) and hippocampal neurons (F) (two-way

ANOVA with Sidak’s post hoc tests: E, p(interaction)=0.434, F(2,24)=0.86; p(isoform)=0.876,

189

F(2,24)=0.13; p(siRNA)=0.441, F(1,24)=0.61; n=5; F, p(interaction)=0.218, F(2,24)=1.63; p(isoform)=0.970, F(2,24)=0.03; p(siRNA)=0.501, F(1,24)=0.47; n=5). (G,H) Quantitative real- time PCR shows no changes in p110 mRNA expression in primary cortical (G) and primary hippocampal (H) cultures after Pten siRNA transfection (two-way ANOVA with Sidak’s post hoc tests: G, p(interaction)=0.913, F(2, 12)=0.09; p(isoform)<0.001, F(2, 12)=126.10; p(siRNA)=0.768, F(2,12)=0.09; n=3; H, p(interaction)=0.222, F(2, 12)=1.710; p(isoform)<0.001,

F(2, 12)=35.99; p(siRNA)=0.206, F(2,12)=1.79; n=3); N are independent neuronal cultures.

Error bars represent SEM.

190

Figure S2: Mechanisms of p110β regulation may differ in PTEN-deficient cancer cells and

PTEN-deficient primary neurons. Immunoprecipitations of PC3 cancer cells (A) and primary neurons transfected with scrambled or Pten siRNA (B) confirm association of p110β with CRKL in PTEN mutant PC3 cells but did not detect association of p110β with CRKL in Pten deficient primary neurons. We likewise did not detect p110β in CRKL-immunoprecipitates from Pten mutant brain lysates (data not shown). IPs were repeated 3 times. I=IgG, C=CRKL, Ctr=WT control cortex, Scr=Scrambled siRNA, Pten=Pten siRNA. Brackets indicate CRKL-specific signal.

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Figure S3: Protein expression of p110 isoforms remains unchanged in Pten; CamKdel mice. (A-D) Pten; CamKdel mice show a decrease in PTEN expression compared with littermate controls in cortex (A,C) and hippocampus (B,D) (unpaired t-tests: C, ***p<0.0001, t(14)=5.14; n=8; D, ***p=0.0005, t(14)=4.47; n=8). (E,F) Protein expression levels of p110α, β, and δ are unchanged in Pten; CamKdel mice compared with control littermates in cortex (E) and hippocampus (F) (two-way ANOVA with Sidak’s post hoc tests: E, p(interaction)=0.36,

F(2,42)=1.05; p(isoform)=0.94, F(2,42)=0.06; p(genotype)=0.89, F(1,42)=0.02; n=8; F, p(interaction)=0.73, F(2,24)=0.32; p(isoform)=0.71, F(2,42)=0.34; p(genotype)=0.026,

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F(1,42)=5.34; n=8. N denotes individual animals. Error bars represent SEM. Representative western blots are shown in A and B.

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Figure S4: Oral GSK6A administration at 5 mg/kg in peanut butter reduces elevated AKT phosphorylation in Pten; CamKdel mice. Western blots showing that oral administration of

GSK6A in peanut butter for one week reduces AKT phosphorylation to approximately control littermate levels at a dose of 5 mg/kg but not 2.5 mg/kg. Doses were recommended by

GlaxoSmithKline after in-house assessment of oral dosing. Further analyses of the effects of oral GSK6A on signal transduction including quantifications are shown in Figure 4.

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Figure S5: Pten; CamKdel mice have increased S6 Kinase phosphorylation, but not S6 phosphorylation in cortex and hippocampus. (A,B) Representative western blots of S6K and

S6 phosphorylation in cortical (A) and hippocampal (B) lysates of a Pten; CamKdel mouse and a littermate control after one week of daily oral administration of GSK6A or vehicle. (C,D)

Phosphorylation of S6 kinase is overall increased in cortex and hippocampus of Pten; CamKdel mice, and GSK6A treatment reduces S6 kinase phosphorylation independently of genotype in hippocampus (D), but not in cortex (C) (two-way ANOVA with Tukey’s post hoc tests: C, p(interaction)=0.641, F(1,24)=0.22; p(treatment)=0.228, F(1,24)=1.53; p(genotype)=0.012,

F(1,24)=7.34; D, p(interaction)=0.652, F(1,24)=0.21; p(treatment)=0.035, F(1,24)=5.02; p(genotype)=0.0003, F(1,24)=18.27). (E,F) No effects of genotype or treatment on S6 phosphorylation were detected in either cortex (E) or hippocampus (F) (two-way ANOVA with

Tukey’s post hoc tests: E, p(interaction)=0.454, F(1,24)=0.58; p(treatment)=0.155,

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F(1,24)=2.16; p(genotype)=0.898, F(1,24)=0.02; F, p(interaction)=0.058, F(1,24)=3.97; p(treatment)=0.241, F(1,24)=1.45; p(genotype)=0.274, F(1,24)=1.25). No differences in pairwise comparisons were detected; n=7 per group. Error bars represent SEM.

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Figure S6: GSK6A administration may reduce gamma EEG power and frequency of EEG spikes and spike trains in Pten; CamKdel mice. (A,B) Power analyses show increased gamma/total power ratios over time during the day (A) and night (B), which may be reduced with

GSK6A treatment (mixed effect analysis with Sidak’s post hoc tests: A, p(day)=0.277,

F(10,38)=1.28; p(treatment)=0.485, F(1,4)=0.59; p(day x treatment)=0.484, F(10,38)=0.97,

GSK6A: n=3 all days, vehicle: n=3 all days except for days 4 and 5 (n=2); B, p(night)=0.429,

F(10,36)=1.04; p(treatment)=0.516, F(1,4)=0.51; p(night x treatment)=0.473, F(10,36)=0.99,

GSK6A: n=3 all nights except for night 11 (n=2), vehicle: n=3 all nights except for nights 2, 4 and 5 (n=2)). (C,D) Number of spikes during a 2-hour period during the day (C) and night (D) in

Pten; CamKdel mice is increasing over time, which may be reduced by GSK6A treatment

(mixed effect analysis with Sidak’s post hoc tests: C, p(day)=0.061, F(10,33)=2.04; p(treatment)=0.467, F(1,4)=0.64; p(day x treatment)=0.0.067, F(10,33)=2.00, **p=0.002;

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GSK6A: n=3 all days except for days 10 and 11 (n=2), vehicle: n=3 all days except for days 4,

5, 9-11 (n=2); D, p(night)=0.206, F(10,32)=1.45; p(treatment)=0.732, F(1,4)=0.14; p(night x treatment)=0.726, F(10,33)=0.69, GSK6A: n=3 all nights except for nights 10 and 11 (n=2), vehicle: n=3 all nights except for nights 3-5,9-11 (n=2)). (E,F) Number of spike trains during a 2- hour period during the day (E) and night (F) in Pten; CamKdel mice is increasing over time, which may be reduced by GSK6A treatment (mixed effect analysis with Sidak’s post hoc tests:

E, p(day)=0.772, F(10,33)=0.64; p(treatment)=0.86, F(1,4)=0.04; p(day x treatment)=0.674,

F(10,33)=0.75, GSK6A: n=3 all days except for days 8-11, vehicle: n=3 all days except for day 5

(n=2); F, p(night)=0.328, F(10,35)=1.19; p(treatment)=0.795, F(1,4)=0.08; p(night x treatment)=0.961, F(10,35)=0.35, GSK6A: n=3 all nights except for nights 9-11 (n=2), vehicle: n=3 all nights except for nights 4 and 5 (n=2)). Box represents the treatment period. Error bars represent SEM. All analysis was performed by Dr. Durgesh Tiwari, PhD.

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GSK2702926A IC87114 BYL719 p110α 794 nM >100 μM 5 nM p110β 0.2 nM 75 μM 1200 nM p110γ 1000 nM 29 μM 250 nM p110δ 7.9 nM 0.5 μM 290 nM

Table 1: Isoform-selectivity of GSK2702926A, IC87114, and BYL-719. IC50 for each compound is listed, showing the high selectivity of each compound to only one class 1A isoform.

Data for GSK2702926A were taken from (Gross et al., 2019), data for IC87114 are from (Sadhu et al., 2003), and data for BYL719 are from (Furet et al., 2013).

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Mouse Allocation N Complete 16 Death Prior to Surgery 5 Death Prior to Treatment 13 Death During Treatment 3 Controls (Survive) 12 Controls (Fatal) 1 Mouse Allocation N

Table 2: Mortality throughout EEG studies in Pten; CamKdel mice. Shown are number of

Pten; CamKdel mice (top 4 rows) and control mice (bottom two rows) that completed the study as well as those who died during various stages of the study.

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Chapter 5: Discussion

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The collective work presented in this thesis examines PTEN deficiency in mouse models for high grade gliomas and autism with an epilepsy co-morbidity. In Chapter 2, we introduced a novel mouse model for high grade glioma with PTEN deficiency and subjected the mice to therapy which inhibits the PI3K pathway. The original hypothesis was PI3K inhibition would decrease cell proliferation and increase apoptosis; however, we found only a subset of these tumors had a favorable response. Preliminary assessment of these tumors shows the tumors that are not responsive to PI3K inhibition have more stem cell-like or progenitor cells as compared to their more differentiated counterparts. In a second study, we introduced a pediatric mouse model for high grade glioma with two driving mutations from the PI3K pathway, including

PTEN and performed a series of experiments assessing PI3K pathway inhibition in the model.

In a third study, we applied a therapeutic strategy which has shown promise in PTEN-deficient cancers to a mouse model for PTEN-deficient autism and found a rescue or partial rescue of phenotypes.

The Connection between Cancer and Autism

In 1997, cancer geneticist Dr. Charis Eng observed a connection between individuals diagnosed with Cowden syndrome (a PTEN-linked disorder characterized by benign growths and a high lifetime rate of cancer) and a high frequency of autism in families with Cowden syndrome patients (Butler et al., 2005; Opar, 2017; Yehia et al., 2020). In a small study based on this observation, 3 of 18 children with autism and macrocephaly had a mutation in PTEN

(Butler et al., 2005). The link between PTEN and autism is so widely accepted now that genetic testing for PTEN mutations is a recommendation for autistic individuals with macrocephaly

(Buxbaum et al., 2007).

Since this connection between PTEN and autism was discovered, additional associations between components of the PI3K pathway, including its regulators, and autism have been found.

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Insulin-like growth factor-I (IGF-I) is an upstream regulator of PI3K through its binding to the receptor tyrosine kinase (RTK) IGF receptor-1 (Laviola, Natalicchio, & Giorgino, 2007). It has been shown in several studies that IGF-1 levels are lower in both cerebrospinal fluid and urine of children with autism as compared to age-matched controls (Anlar et al., 2007; Riikonen et al., 2006; Vanhala, Turpeinen, & Riikonen, 2001).

Neurofibromatosis type I (NF1) is a tumor suppressor protein which is known to regulate both TSC1 and mTOR (Johannessen et al., 2005). Mutations in the NF1 gene are linked to a high incidence of non-cancerous growths but has an association to cancerous growths in some instances (Zhu, Ghosh, Charnay, Burns, & Parada, 2002). In several studies, it has been demonstrated that individuals with autism have a 100 to 200 fold increase in neurofibromatosis as compared to individuals without autism (Gillberg & Forsell, 1984; Marui et al., 2004; Mbarek et al., 1999).

As previously discussed, patients with tuberous sclerosis complex (TSC) often have autism as a co-morbidity. The TSC proteins (1 and 2) are upstream of and regulators of the mTORC1 complex through suppression of Rheb (Chapter 1, Figure 3) (Inoki, Corradetti, &

Guan, 2005).

Beyond the PI3K pathway, additional pathways associated with cancer have been linked to autism. For example, it is known that individuals with RASopathies have higher incidents of autism as compared to unaffected controls (Adviento et al., 2014). Through NF-1, this pathway is also linked to PI3K signaling. Table 1 includes a summary of pathways that have been associated to both cancer and autism (Wen & Herbert, 2017). There are now at least 43 genes mutually associated with both cancer and autism as assessed by GeneAnalytics, which is a computational approach that incorporates a bioinformatics pipeline developed by GeneCards

(Gabrielli, Manzardo, & Butler, 2019).

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Additional mechanisms beyond specific cell signaling pathways have been noted to be shared between both cancer and autism (Crawley, Heyer, & LaSalle, 2016). Chromatin remodeling genes are dysregulated often, which can result in changes in gene accessibility for transcription. Genome maintenance genes are also aberrantly regulated in both disorders and include cellular processes such as DNA repair and cell cycle control (Crawley et al., 2016).

Finally, transcription factors are found to be commonly dysregulated in both disorders (Table 1)

(Crawley et al., 2016). Transcription factors are vital both in embryonic development and normal cell signaling and alterations can have detrimental effects on gene expression (Latchman,

1996).

Cellular processes and pathways which are aberrantly regulated in cancer and autism, including the PI3K pathway, are often implicated in cellular development. Dysfunction yields changes in cells or a system. In cancer, cell proliferation, cell size, and cell division collectively create a system of dysfunction within an organ or system which facilitates increased and uncontrolled cellular growth as well as invasion of cancer cells either locally to an organ or throughout the body in a process known as metastasis (Zang et al., 2014; Zheng et al., 2003).

In autism, these same cellular processes are often dysregulated, but the consequences are mostly milder and include increases in neuron size and neurite outgrowth. These changes create abnormal brain connectivity (Tilot, Frazier, & Eng, 2015). Proper connectivity is vital for how the brain intakes and processes information. Changes in connectivity can lead to a multitude of dysfunctions, including epilepsy and autism.

The molecular link between cancer and autism has proven beneficial in the study of autism, as there has been reported success in the utilization of cancer therapy in neurological disorders such as TSC (Krueger et al., 2013). This type of therapeutic approach is the basis of work performed in Chapter 4. Here, we utilized p110β inhibition in PTEN-deficient, neuron- specific mouse models. Given the additional druggable targets that are now collectively

204 associated with both cancer and autism, this work argues in favor of assessing specific, molecular-based therapies developed to target cell signaling aberrations in cancer for parallel subsets of autism spectrum disorders.

PTEN Regulation and its Implications in this Research

As extensively discussed in Chapter 1, PTEN regulation is a very complex set of biological processes. Many of the ways in which PTEN is regulated, such as transcriptional, translational, and post-translational regulation, are normal physiological processes. These processes maintain PTEN levels in a healthy range; however, if there is aberrant regulation of any of the pathways or biological processes which control PTEN expression, this would ultimately lead to altered PTEN function. Defects in PTEN regulation can be caused by gene mutations, epigenetic changes, or environmental influences (Bermudez Brito, Goulielmaki, &

Papakonstanti, 2015). Given this knowledge, it is possible that the quantity of patients that are

PTEN-deficient, in both cancer and autism, is higher than the current estimates. This argues that there could be a larger population which would benefit from understanding how PTEN dysregulation contributes to disease, including but not limited to brain tumors and PTEN-linked autism.

There is potentially a larger population of patients with aberrantly regulated PTEN but there are several factors which are yet to be elucidated. First, what type of diagnostics would have to be implemented in order to assess patients with PTEN deficiency not caused by a mutation? In cancer, as seen in Chapter 3 of this work, a sample of the tumor is generally submitted for assessment for common cancer mutations, including PTEN. As with cancer,

PTEN is often assessed if a patient presents with signs of neurological developmental delay or in individuals with a patient history of PTEN-associated disease. In both cancer and autism, screening for the regulators of PTEN is difficult if not impossible.

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Finally, it is not well understood how the variance in PTEN regulation may create different disease phenotypes. To elaborate, when PTEN deficiency is a result of a mutation, we know, as discussed in the introduction, that different mutations yield different downstream signaling consequences (Spinelli, Black, Berg, Eickholt, & Leslie, 2015). Do changes in post- translational modifications of PTEN produce the same downstream signaling effects as changes in its transcriptional regulation if both result in PTEN deficiency? Answering these questions would be a daunting task and not likely to be accomplished in the near future. There are both in vitro and in vivo examples of models which have been utilized to elucidate many of the pathways that regulate PTEN, as discussed in the introduction. A detailed analysis of the downstream effects of the various post-translational or transcriptional factors that can alter

PTEN function and/or expression, through a series of experiments such as western blots, would be a first step to assess the downstream results. These could be similar to western blot analyses as performed in Chapter 4, Figure 4 and Figure Supplemental 5. As a general example, Bmi-1 regulation of PTEN was discussed in the introduction of this thesis (Song et al.,

2009). The nasopharyngeal epithelial cell culture lines utilized by Song, et al are commercially available, as is the shRNA. Bmi-1 depleted epithelial cultures could be collected and subjected to western blot in order to assess PI3K downstream signaling (Song et al., 2009).

High Grade Gliomas: Implications of Dissertation Research

In the background for Chapter 2, I discussed the dismal prognosis for high grade gliomas. Despite extensive research for over 20 years, survival has not significantly improved.

In both research projects that assess high grade gliomas, we took a pathway-specific therapeutic approach targeting PI3K, which is known to be dysregulated in 88% of HGGs

(Langhans et al., 2017; Li et al., 2016). Interestingly, in Chapter 2, we found that a subset of

QKO tumors were responsive to inhibition of PI3K and its downstream effectors (either as a singular therapy or a combination therapy) while other tumors did not respond. The preliminary

206 work included in Chapter 2 suggests that tumors with a higher quantity of stem and progenitor cells are less responsive as compared to tumors that consist of differentiated cells. As shown in

Chapter 2, spontaneous tumors generated in the QKO model can genetically mimic each of the gene expression subgroups of glioma (Olar & Aldape, 2014).The basic classification done through immunohistochemistry (IHC) in Chapter 2 suggests that responsive tumors belong to one of the HGG subgroups but this needs to be thoroughly elucidated with RNA-seq.

The cancer stem cell hypothesis is based upon an observation that most tumors retain a small subset of cells which have stem cell characteristics, primarily the ability to self-renew, remain in an undifferentiated state, and produce most of the cell types found within the tumor

(Moharil, Dive, Khandekar, & Bodhade, 2017). These initiating cells retain characteristics that make them more resistant to many forms of chemotherapy (Moharil et al., 2017). Many conventional therapies for cancer target cells that are rapidly dividing as a majority of cancer cells do (Bagnyukova et al., 2010). In contrast, cancer stem cells remain quiescent and thus often less susceptible to therapy (Hermawan & Putri, 2020). These cells are therefore able to repopulate the tumor while maintaining a small population of stem cells.

In Chapter 3, we discussed the lack of model systems that accurately depict pHGGs. As part of this thesis work, we were able to successfully generate a primary tumor orthotopic model that better mimics key characteristics of pHGG. Our work suggests that tumors collected during biopsy were more likely to successfully transplant than those collected at the time of autopsy.

No additional assessment was performed in order to learn what, if any, specific characteristics are more conducive to successful implantation, but this information is helpful for scientists attempting to generate HGG models.

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Potential Utility of Our Findings for High Grade Glioma Research

QKO Mouse Model Research

What molecular qualities make a subset of QKO tumors responsive to PI3K pathway inhibition while other QKO tumors are non-responsive? To continue to analyze this question, I propose the sampling of QKO tumors after PI3K (with or without mTOR) inhibition and assessment through RNA-seq in order to determine if there is a correlation in the gene expression subgroup and the responsiveness of the tumor to PI3K inhibition. For this work, I would do one week of PI3K pathway inhibition with the PI3K pathway inhibitors utilized in

Chapter 2. After this week, I would sacrifice the mice and collect the brain. One half of the brain would be used for IHC (for the purpose of grouping tumors by responsiveness) and one half would be used for RNA-seq, dissecting tumor material from normal material. Given the IHC results which led to the suggestion that more differentiated tumors are more responsive to PI3K inhibition, it is likely that a genetic subtype or multiple subtypes are responsive to PI3K inhibition while other subtypes are not responsive to PI3K inhibition. RNA-seq is the standard which is utilized in order to group gliomas into their genetic subtype. To speculate, given that both p53 (which was introduced as a PTEN regulator) and PTEN are dysregulated in this model, the PI3K pathway is consistently dysregulated in QKO tumors. We also observed, through IHC, that QKO tumors treated with vehicle have consistently higher levels of downstream effectors pAkt and pS6 than tumors treated with PI3K inhibitors, suggesting our therapy was efficacious at the molecular level. I think the variance seen in tumor response is a product of pathways found to regulate the stem and progenitor population. It is known that the

Notch pathway is upregulated in one subset of QKO tumors (unpublished data but partially introduced in Chapter 2, Figure 1). Notch signaling is known to regulate glioma stem cells; therefore, I speculate this could be one of the pathways differentially regulated in the subtypes

(Hovinga et al., 2010; Ying et al., 2011).

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In Chapter 2, we demonstrated that the QKO tumors respond to therapy currently used in humans (radiation and temozolomide) in a manner similar to human tumors. Therefore, if

RNA-seq were to identify subsets of responsive tumors characterized by a certain transcriptomic profile, this may be translational to a group of HGG patients. To elaborate, the genetic subtype of tumors could be assessed at time of biopsy and this subtype could potentially help predict what patients may be responsive to PI3K pathway inhibitors. It is worth noting that PTEN deficiency is not sufficient to determine if PI3K pathway inhibition is efficacious (Chalhoub & Baker, 2009). If RNA-seq data does not reveal a tumor subset or group of subsets which respond favorably to PI3K pathway inhibition, this data would still prove beneficial, as it would be significantly more comprehensive than what we were able to do with immunohistochemistry. This could; therefore, provide a unique genetic signature which could be identified in biopsy samples and then considered when choosing a therapeutic strategy.

Dependent on what is found in this proposed study of QKO tumors, my next anticipated experiment would be to utilize human tumors and perform a similar RNA-seq analysis in order to assess if our work in the QKO model is translational. The tumor sample, taken at the time of resection, could be analyzed for RNA-seq. There are currently ongoing and recruiting clinical trials which are utilizing numerous inhibitors which target PI3K signaling, as per

ClinicalTrials.gov. As already illustrated, there are completed solid tumor studies which utilize

PI3K pathway inhibition; some of which may have resected tumor material. Tumor material taken at either surgical resection or autopsy could be utilized to assess the genetic subtype of tumors. In many instances, this subgroup may be known. Given the high rate of surgically resected tumors, these studies could be easily powered for strong statistical analysis. This sequencing data could be compared to the patient’s reaction, as assessed by both tumor regression, survival, and IHC (KI67 and cleaved caspase-3) of resected material. If our ongoing hypothesis is correct in this set of experiments, that the tumor response to PI3K pathway

209 inhibition is defined by the genetic subtype of the brain tumor, this would be a strong favorable argument to design a clinical trial which stratifies patients by their tumor gene expression subtype and specifically administer PI3K inhibitors to specific genetic subtypes.

This strategy could be further expanded to other genetic subtypes. For example, it is known that there are brain tumors within the genetic subtype defined as proneural that have aberrant Notch signaling (Saito et al., 2014). There are Notch specific inhibitors which could be utilized in the proneural subset.

In addition to this work and taking into consideration the hypothesis that many brain tumors contain stem cells, I would also propose an additional set of experiments in the QKO mouse model which integrates the therapies utilized in Chapter 2 but also additional therapies aimed at targeting the stem cell niche of the tumor.

There are a small number of approaches to target the stem cell component of brain tumors currently being examined. One of the more progressed (as per clinical trial data availability) is vaccination with dendritic cells and RNA derived from tumorspheres. Dendritic cells are considered the most efficient type of antigen presenting cell in the immune system

(Vik-Mo et al., 2013). Stem cell cultures (tumorspheres) have been created from individual patients and RNA was isolated and amplified from these cultures. Dendritic cells were transfected with the amplified sample and injected intradermally (ID) (Vik-Mo et al., 2013). Table

2 illustrates a hypothetical experiment in order to study our previous pharmacological strategy in conjunction with stem cell specific therapy.

The initial readouts for such a study would be KI67 to assess changes in tumor cell proliferation and cleaved caspase-3 to assess apoptosis, as utilized in Chapters 2 and 3. My hypothesis is tumors subjected to a combination therapy which includes PI3K pathway inhibition and dendritic cell injection, to target the stem cell population, would

210 significantly decrease proliferation and increase apoptosis. Assessment of tumor responsiveness could be performed using the same methods previously outlined in Chapter 2 and Chapter 3.

Pediatric HGG Modeling and Future Research

If this project were allowed to continue, the first experiments would be the previously mentioned additional PI3K therapy administration to obtain sufficient numbers for statistical analysis. If sufficient mice were available, it would be interesting to also assess the therapy administered to the donating patient and compare treatment responses of the utilized therapy and our novel therapeutic strategy given to our mouse model.

An exciting project based on findings of both Chapters 2 and 3 would be to collect tumor samples at the time of the initial biopsy and implant the tumors immediately using the same technique as described in the Chapter 3 pediatric brain tumor model. RNA-seq would be utilized at this time (on the donating tumor) in order to determine the genetic signature. Once the tumor was propagated in mice and given the genetic subtype which would have already been acquired, a small study which would utilize pathway specific therapy as well as standards of care could be performed in order to assess an individual’s response to therapy. This could potentially predict patient response and find a population that may respond more favorably to

PI3K therapy (Krepler et al., 2016; Lodhia, Hadley, Haluska, & Scott, 2015; Scott et al., 2015).

An example study is listed in Table 3. Please note this is an example based on a hypothetical tumor that has been deemed potentially responsive to PI3K inhibitor responsive. BMK120 is a pan PI3K inhibitor and represents pathway specific inhibition. Temozolomide represents a current standard of care but the dosing protocol used here is different, as it is the same as the protocol previously used in this work (Chapter 2) (Olson et al., 2010). The approach used to construct these studies could potentially change, dependent on the preceding study data.

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This type of experiment would require a larger donation of tumor as compared to what is normally obtained. If that is not possible, the experiment could be done after the first passage from the original mouse implant to the second mouse implant. Mice would be monitored with imaging in order to assess when the tumor is established in the model (McConville et al., 2007).

Given the promising response, though not adequately powered for statistics, in Chapter

3, I hypothesize that PI3K inhibition would significantly decrease tumor growth as compared to vehicle treatment in tumors with confirmed PTEN mutations. Based on our limited sample size, it is difficult to speculate how PI3K inhibition would respond in comparison to current therapeutics, such as the alkylating agent temozolomide.

Limitations and Potential Implications of High Grade Glioma Research

The QKO mouse which was utilized in Chapter 2 is an accurate model for the purpose of studying HGG; however, the mice frequently have multiple tumors and the tumors have been shown to vary molecularly within a mouse (data now shown). Therefore, within a singular mouse, you may have a tumor which is responsive to therapy and another tumor that is unresponsive. This makes survival of the mouse difficult to interpret.

The multiple tumors, in conjunction with the aggressive growth of the tumor(s) yields a short time to moribundity. Within a week, mice may go from asymptomatic to moribund. Here, we used mice displaying mild signs of illness and; therefore, some mice died before the completion of the experiments. This could potentially skew the final data if there was a certain subset of mice, as defined by the type or types of tumors present. It is not known what factor(s) allow some mice to easily complete the treatment regimen while other mice do not and it is not understood how this affects data interpretation. Potential causes could be related to the genetic subtype of the tumors or it could be another factor such as location of the tumor(s) in the brain.

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In Chapter 3, I introduced a novel mouse model for the study of pHGG. This model could be highly valuable as there is a lack of modeling systems which properly mimic pHGG; (Holland,

2001) however, there are many considerations when assessing this model. The time from tissue resection until it is delivered to the lab is expected to affect cell viability and should be kept as short as possible. The sample is then minced and processed in order to prepare the sample to be able to pass into and out of a Hamilton syringe. These processes likely reduce cell viability.

What we do not understand about this process is if certain cells within the heterogeneous sample have a propensity for cellular death as compared to other cells within the sample. If so, it is likely that a vital component of the donating tumor is no longer present. Above this, each serial passage of the tumor goes through the same process which may further select for a certain portion of the tumor.

Additionally, as mentioned in Chapter 3, the tumor environment is different in the mouse compared with the donating patient, including stromal components and the immune system.

Through Foundation One™ sequencing (Chalmers et al., 2015) and immunohistochemistry, we were able to demonstrate that these tumors maintain characteristics of the donating tumor; however, the stromal characteristics of the tumor are likely very different which may affect tumor response. This is often a concern with any orthotopic model developed in cancer research.

Additionally, the mice utilized in these models are immunocompromised. Given that the immune system is known to play an important role in cancer initiation, growth, and treatment, this factor must be taken into account when using the model to assess both tumor characteristics and treatment (Gonzalez, Hagerling, & Werb, 2018).

In conclusion, the limitations of this tumor model are equal to the concerns of any patient-derived orthotopic tumor and these limitations should be taken into consideration. These models are still disease-relevant and create a germane way to assess novel therapy in a tumor microenvironment.

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Implications of p110β Inhibition in PTEN-deficient Autism

Outlooks of PTEN Deficiency Research and Potential Therapy

The reversal of fundamental autism clinical symptoms has not been accomplished to date, likely due to complex and diverse etiologies. Often, current therapies utilized for autism target specific clinical presentations or comorbidities of autism but are not disease mechanism- targeted and thus unable to reverse the defining clinical deficits (Sathe, Andrews, McPheeters,

& Warren, 2017; Weitlauf, Sathe, McPheeters, & Warren, 2017). This therapeutic approach is favorable for the treatment of certain symptoms, but the molecular underpinnings of the disorder are untreated; therefore, additional clinical symptoms are either untreated or additional therapy is required. Adjunct therapy may be expensive, dangerous, and ineffective (Table 4) (Brasic et al., 1994; Brodkin, McDougle, Naylor, Cohen, & Price, 1997; Buitelaar, van der Gaag, & van der

Hoeven, 1998; Erickson et al., 2014; Ghaleiha et al., 2013; Gordon, State, Nelson, Hamburger,

& Rapoport, 1993; Guastella et al., 2015; Hardan et al., 2012; Harfterkamp et al., 2012;

Hollander et al., 2007; Hollander et al., 2005; Hollander et al., 2012; King et al., 2009; McDougle et al., 1992; Murray, 2010; Posey, Guenin, Kohn, Swiezy, & McDougle, 2001; Scahill et al.,

2006). Table 4 illustrates therapies that are currently being utilized to assuage symptoms or comorbidities of autism. Some of these therapies have severe side effects. Additionally, some of these compounds cannot be administered in conjunction with other therapies.

In this work, we utilized an approach aimed at a specific disease mechanism in neuron- specific, PTEN-deficient mouse models. P110β inhibition has been found to be efficacious in

PTEN-deficient cancer (Jia et al., 2008; Wee et al., 2008). Our approach of inhibiting the PI3K catalytic isoform, p110β, in this mouse model of PTEN-associated neurodevelopmental disorders normalized increased protein synthesis and PI3K signaling, reduced seizures, and reduced certain aspects of macrocephaly. We did not report any side effects from the dosing regimen and mice did not lose any weight, suggesting this approach may be safe in patients.

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As discussed in the introduction of this thesis, there are additional therapies, which have been investigated in both animals and humans, that inhibit the PI3K pathway, generally either

PI3K or mTOR (Komiya et al., 2019; Ljungberg, Sunnen, Lugo, Anderson, & D'Arcangelo, 2009;

Maire et al., 2014; Sunnen et al., 2011; Zhou et al., 2009). This work has reported favorable outcomes; however, inhibition of p110β in our studies did not yield the weight loss which has been noted in mTOR inhibition studies performed in mice (Bitto et al., 2016; Johnson et al.,

2015). Pan PI3K inhibitors have also created severe body weight loss and additional clinical concerns with long-term use (Hanker, Kaklamani, & Arteaga, 2019). MTOR inhibition is several steps downstream of PTEN; therefore, the depletion of PIP2 and the accumulation of PIP3 will still occur. As presented in the introduction, PIP2 plays many important roles in endocytosis and exocytosis and it is likely to still be diminished with mTOR inhibition (Huang, 2007). Similarly,

PIP3 is probably still found in higher proportions than cells with normal PTEN levels. Thus, PIP3 would still be actively recruiting and activating increased quantities of downstream effectors such as Akt (Maehama & Dixon, 1998). Our strategy of inhibiting p110β should maintain levels of PIP2 and PIP3 in levels similar to cells unaffected by PTEN deficiency; however, we have not confirmed the levels of PIP2 and PIP3 as compared to controls to date.

Assessing Potential Disease Modifying Effects and Underlying Mechanisms of p110β

Inhibition

One aspect of the work done in Chapter 4 that is not well understood is if p110β inhibition is disease modifying or if it is symptomatic therapy (Cummings, 2009). Disease modifying is to correct the underlying mechanism of the disease which in turn, halts the disorder while symptomatic therapy simply treats a symptom(s) of the disease, leaving the underlying disorder intact. The best way to address this issue is to administer therapy, discontinue the therapy, and allow the animals to stay on study after p110β inhibition is discontinued. The high

215 mortality of the Ptenfl/fl; CamK Cre+ mouse model made this difficult, if not impossible to address in our studies.

The best way to initially address this issue would be to utilize the Ptenfl/fl; Gfap+ mouse model, which survives a 4-week drug dosing regimen. Because we do know from the literature that this model dies prematurely, beginning at approximately nine weeks, I would propose a daily therapy administration which starts at 3 weeks of age and ends at 7 weeks of age (Kwon et al., 2001). This time point should circumvent losing mice to this premature death which could potentially alter the data. Phenotypes such as signal transduction and macrocephaly would be assessed two weeks after treatment has concluded. I hypothesize that, given our lab’s drug washout studies performed in a FXS mouse model which show a decrease in Akt phosphorylation at one hour post dose administration which is no longer evident at 24 hours

(Gross et al., 2019), normalized cell signaling after p110β inhibition would return to increased levels in mutant mice compared to control littermates. Additionally, adaptive resistance is a common mechanism which restores the PI3K pathway and this monotherapy would not inhibit any of the mechanisms that could potentially reactivate PI3K signaling. One example of adaptive resistance is the reactivation of the downstream effector S6K1 which subsequently hyperactivates Akt (O'Reilly et al., 2006).

Prevention studies have demonstrated that when mTOR inhibition is administered before mice reach adulthood, certain aspects of the development of macrocephaly are able to averted

(Zhou et al., 2009). Thus, I hypothesize that p110β inhibition given for four weeks in presymptomatic mice would have a disease modifying effect on soma size and abnormal neurite outgrowth. This hypothesis is highly dependent on the age of the mice (Zhou et al.,

2009). Structural changes would be corrected by a long dosing regimen given early and this corrects or partially corrects macrocephaly.

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Another exciting experiment that could be performed in this mouse model would be to assess the seizure phenotype utilizing the same age and dosing protocol as presented above

(Kwon et al., 2001). The seizure phenotype has been documented in the literature; however, we have not been able to confirm the phenotype to date (Backman et al., 2001). Because of the brain morphological phenotypes that are likely to be corrected by early dosing, I hypothesize that there would be a disease modifying effect on seizures.

Another experiment which could be performed in order to address this issue would be a survival study similar to what is illustrated in Chapter 4, Figure 3. I would propose daily IP injection of either GSK6A or vehicle in Ptenlfl/fl; CamKII-Cre mice beginning at 3 weeks of age, when these mice are presymptomatic. I would administer 14 days of therapy, cease therapy, and monitor daily for signs of moribundity. If p110β inhibition has disease modifying effects in the context of brain-specific PTEN deficiency, mutant mice treated with GSK6A would survive significantly longer than mutant mice treated with vehicle. I hypothesize that p110β inhibition would have disease modifying effects on survival as compared to vehicle treated mutant mice. As previously stated in relation to the first presented disease modifying experiments, the time in development would be highly important. This time point is intended to prevent some of the structural abnormalities that occur in the brain during development. If these neuronal architecture abnormalities are not corrected, I would expect there would be no disease modifying effect and you would not see any differences in survival. It is worth noting that this experiment may not be an option if the mice die during this early time point due to the stress of being handled. It is unclear when this phenotype begins.

To address the mechanism of why p110β inhibition rescues or partially rescues many phenotypes in neuron-specific mouse models of PTEN deficiency, we performed a series of western blots in which we quantified protein expression of p110α, β, and δ with the hypothesis that in PTEN deficiency, there would be an increase in p110β as compared to controls with

217 normal PTEN levels. In all three isoforms, there was no significant difference in protein expression. These experiments rule out one of the explanations for the reversal of phenotypes reported in Chapter 4.

In PTEN-deficient cancer cells, there is a proposed mechanism to describe preferential activation of p110β in PTEN deficiency. Zhang et al propose a model that includes a protein interaction between the adaptor protein CRKL and p110β. This interaction is not found with other PI3K isoforms (Zhang et al., 2017). In the model, CRKL is in a complex with p110β and the scaffolding protein p130Cas. Effector proteins within this complex ultimately activate Rac signaling. Activation of Rac signaling further perpetuates PI3K signaling, yielding a positive feedback loop (Zhang et al., 2017). The importance of Rac signaling in the context of the brain will be discussed again below. This interaction does not occur when PTEN is present, as PTEN negatively regulates two proteins in this complex, FAK and Src, which alters the conformation of p130Cas so that p110β is unable to bind to the complex.

In order to assess if there is a similar interaction between CRKL and p110β in the brain, we performed a series of immunoprecipitations in primary cortical neurons and Ptenfl/fl; CamK

Cre+ cortical lysates. The interaction was not found in either of our models. We utilized a PTEN- deficient cancer cell line (PC3) as a control and the interaction was found in this cell line.

In both of our modeling systems utilized for IP experiments PTEN is decreased yet present. For example, PTEN western blots in the Ptenfl/fl; CamK Cre+ mouse model show approximately 20% decrease in PTEN as compared to the control littermates (quantifications obtained from Chapter 4, Figure S3 western blots). In primary neuronal cultures, we report 25-

35% (cortical and hippocampal neurons respectively) decrease in PTEN after treatment with

Pten siRNA (Figure S1) compared with scrambled siRNA. The tumor cell line used to assess p110β and CRKL interactions are PTEN-null. Given the proposed mechanism previously described, it is plausible that this CRKL and p110β interaction is also evident in our models but

218 because PTEN is still present, it may be in proportions too low to detect with immunoprecipitation and western blot. The easiest way to continue this assessment would be to utilize the primary neuronal cultures introduced in Chapter 4, Figure 1 of this work and increase the siRNA concentrations in order to attempt a nearly complete knock down of Pten. Prior to this experiment, we would need to validate the level of knock down to knock out which is feasible in our neuronal cultures before we induce cellular death. If this cell culture technique proves unsuccessful, we could attempt to culture cells from one of our PTEN-deficient mouse models.

Alternatively, I would perform a series of mass spectrometry experiments in addition to the previously described experiment in order to find proteins that preferentially bind to each of the isoforms. To perform the original mass spectrometry experiments in the work describing the interaction between CRKL and p110β, Zhang et al. utilized human embryonic kidney cells (HEK

293 STF) and overexpressed p110α and β (Zhang et al., 2017). I would utilize the same cell line and overexpression protocol as a control and utilize overexpression of p110α, β, and δ in primary mouse neurons. I would use primary neurons instead of Ptenfl/fl; CamK Cre+ and

Ptenfl/fl; CamK Cre- brain lysates because a higher concentration of siRNA could be utilized for a higher level of Pten knock down if needed.

These experiments would not only test if CRKL binds to p110 isoforms in primary neurons, but may identify many other protein interactions specific to each of the class IA catalytic isoforms. It is possible that due to the differences in cell growth and division seen in cancer cells, the interaction is not present in differentiated cells such as neurons. The proposed mass spectrometry analyses could identify other, neuron-specific, unique protein interaction partners of p110β that may explain why p110β drives PTEN deficiency-mediated upregulation of the PI3K pathway in neurons.

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The Connection of PI3K Isoforms and Neurological Disorders

The introduction to this work is focused on PTEN, as it is the common factor in projects conducted in both labs. Another interesting facet yet to be fully discussed are the class I catalytic isoforms of PI3K cell signaling. There are four class I catalytic isoforms that can mediate PI3K activity: p110α, β, δ, and γ. These isoforms differ in structure, regulatory subunits, and function. They are known to have both overlapping and unique functions in both the CNS and throughout the body (Foukas, Berenjeno, Gray, Khwaja, & Vanhaesebroeck, 2010;

Vanhaesebroeck, Guillermet-Guibert, Graupera, & Bilanges, 2010).

Only briefly in Chapter 4 has it been introduced that these isoforms have been associated with specific neurological disorders. This may offer insight into the underlying mechanisms driving multiple neurological disorders and is potentially beneficial as isoform- specific forms of inhibition have been manufactured for cancer (Thorpe, Yuzugullu, & Zhao,

2015). With isoform-specific inhibition, other cellular processes mediated by PI3K signaling are not inhibited, reducing the potential for side effects. Below, we discuss some of the neurological disorders linked to individual PI3K catalytic isoforms.

P110α has been associated with Alzheimer’s disease, megalencephaly, hemimegalencephaly, and epilepsy (Bosco, Fava, Plastino, Montalcini, & Pujia, 2011; T. H. Kim,

Petrou, & Reid, 2014; J. H. Lee et al., 2012; Riviere et al., 2012). These disorders are associated with both cellular enlargement and cell degeneration, interestingly. P110α is unique in the class I PI3K family as a regulator of insulin signaling (Sopasakis et al., 2010). In the brain, insulin signaling is essential for synaptic development and plasticity (C. C. Lee, Huang, & Hsu,

2011; Zhao & Alkon, 2001).

In Chapter 4, we found that p110α inhibition yielded a reduction to control levels in Akt phosphorylation in hippocampal but not cortical primary neurons (Chapter 4, Figure 1). This

220 reduction warrants in vivo assessment; however, the p110α inhibitor used in Chapter 4 does not cross the blood brain barrier (Elkabets et al., 2015). There are no commercially available p110α inhibitors that penetrate the blood brain barrier. This is a limiting factor in the continued assessment of p110α inhibition in the context of neurological disorders, including but not limited to PTEN deficiency in the brain. Direct injection of a p110α inhibitor into the brain ventricles of

PTEN-deficient mice could be used in order to further assess the effects of p110α inhibition in the context of PTEN deficiency. Moreover, inhibition of p110α could have detrimental side effects on insulin signaling in the brain or throughout the body which would make it potentially an unfavorable therapy (Sopasakis et al., 2010).

P110β has several unique characteristics, from its activation to its downstream effectors which may individually or collectively explain why its inhibition, as compared to p110α and δ, rescues multiple phenotypes in the here employed PTEN-deficient, neuron-specific mouse models. P110β is the only PI3K isoform activated by Rab5 and Rac signaling (Fritsch et al.,

2013; Kurosu & Katada, 2001). Rab5 regulates early endosome uptake and fusion. Its upregulation has been shown in the early development of disorders associated with cognitive impairment, including Alzheimer’s disease (Ginsberg, Alldred, et al., 2010; Ginsberg, Mufson, et al., 2010). Rac signaling is considered a master regulator of actin cytoskeletal organization and considered vital for neuronal survival through its regulation of pro-survival proteins Bcl-2 and

Bcl-xL (Bai, Xiang, Liang, & Shi, 2015; Stankiewicz & Linseman, 2014). Within the Rac signaling family of proteins, both Rac1 and Cdc42 have been shown to interact and stimulate p110β after activation of G protein-coupled receptors (Fritsch et al., 2013). To a lesser extent, RhoA and

RohG have also been shown to interact with p110β (Dbouk, 2015). In contrast, additional isoforms of class I PI3K are activated by RAS signaling downstream of RTK activation (Fritsch et al., 2013).

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P110β has been demonstrated to signal preferentially downstream of group 1 metabotropic glutamate (mGlu1/5) mediated protein synthesis (Gross et al., 2015). Depending on the neuronal cell type, it is known that mGlu1/5 mediated signaling can activate multiple kinases in the PI3K pathway, including PI3K, mTOR, and p70 s6 kinase (Harris, Cho, Bashir, &

Molnar, 2004; Hou et al., 2006; Mao & Wang, 2003; G. Page et al., 2006). Reliant upon the context, this activation of downstream effectors may be dependent or independent of the PI3K pathway (G. Page et al., 2006). Group 1 mGlu1/5 protein synthesis is known to serve a vital role in synaptic plasticity, specifically long-term depression, making mGlu1/5 retain a role in learning and cognition (Bellone, Luscher, & Mameli, 2008). This is an important distinguishing factor that may explain the importance of p110β inhibition as compared to the other PI3K class I isoforms in the context of PTEN deficiency in the brain.

P110β has been shown both in our lab and in other labs to be overexpressed in the intellectual disability Fragile X syndrome (FXS) (Gross et al., 2010; Sharma et al., 2010).

Additionally, our lab has utilized the same p110β inhibitor (GSK6A) utilized in Chapter 4 of this work to ameliorate a multitude of aberrant phenotypes in a mouse model of FXS (Gross et al.,

2019). Copy number variations in the gene which codes for p110β (PIK3CB) have been associated with autism (Cusco et al., 2009). This link of phenotypes associated with autism

(repetitive behaviors, communication deficits, and socialization deficiencies) and overactivation in p110β may make p110β a screening tool for autism which could identify a patient population that may benefit from p110β inhibitors.

Another distinguishing characteristic of p110β, which was discovered in the context of cancer, is that when p110β is bound to the regulatory subunit p85, it is not catalytically inhibited.

P85 is a common heterodimer of class IA PI3K catalytic subunits. When bound to p110α, p85 stabilizes and inhibits the catalytic activity of p110α which ultimately inhibits phosphatidylinositol-3, 4, 5-triphosphate (PIP3) production (Dbouk, Pang, Fiser, & Backer,

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2010; Yu et al., 1998). This mechanistic difference in p110β is attributed to its higher level of transformation activity as compared to the other class I isoforms (Thorpe et al., 2015). There could be potential therapeutic implications for the differences in binding, but this is yet to be elucidated. For example, could p85 become a therapeutic target due to its differences in binding to the different PI3K catalytic isoforms?

P110δ has been reported to be aberrantly regulated in schizophrenia (Law et al., 2012).

Two genes considered risk factors in schizophrenia, neuregulin 1 (Nrg-1) and Erb-B2 Receptor

Tyrosine Kinase 4 (ErbB4), have been shown by multiple groups to be the predominant regulators of p110δ (Addington et al., 2007; Law et al., 2006; Stefansson et al., 2002). In a mouse model for schizophrenia with perturbation in Nrg-1, p110δ inhibition has rescued multiple phenotypes including cognitive deficits (Papaleo et al., 2016).

P110γ is the singular isoform in the class IB family of PI3K. This designation is due to the different regulatory subunits that it heterodimers with, p87 and p101 (Vanhaesebroeck et al.,

2010). P110γ has been shown to regulate N-methyl-D-aspartate (NMDA) associated long-term depression (LTD) in the hippocampus (J. I. Kim et al., 2011). An additional link between p110γ and neurological dysfunction has been verified, as the gene which encodes for p110γ has been found to be within the autism susceptibility locus AUTS1 (Kratz et al., 2002). P110γ has not been assessed in this dissertation.

The rescue and partial rescue of multiple phenotypes demonstrated in Chapter 4 utilizing an isoform specific inhibitor, combined with the links of multiple neurological disorders to specific isoforms argues in favor of utilizing an isoform-specific approach for additional neurological disorders which are associated to a PI3K isoform.

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Research Considerations and Limitations for Chapter 4

The mouse models that were utilized in Chapter 4 mimic somatic mutations; however, the role of PTEN in neurological disorders in humans is mainly discussed in the context of germline mutations (Nelen et al., 1997). Most of these mouse models have a well-established seizure phenotype but the literature assessing humans shows a very low occurrence of epilepsy

(Buxbaum et al., 2007; Herman et al., 2007; Lynch et al., 1997; Stein, Elias, Saenz, Pickler, &

Reynolds, 2010; Varga, Pastore, Prior, Herman, & McBride, 2009). Interestingly, as discussed in the mouse model portion of the introduction, there is a model which has been created in order to more closely mimic human PTEN deficiency. These Pten haploinsufficient mice have no reported seizure phenotype, similar as observed in humans (D. T. Page, Kuti, Prestia, & Sur,

2009). It is difficult to fully understand the consequences of the mutation variance of each model, the resulting protein, and what this variance means for research translation.

With this consideration in mind, it is important to note that increased cell signaling downstream of PI3K, including both Akt and mTOR, has been linked to focal cortical dysplasia

(FCD), which leads to intractable epilepsy (J. K. Kim et al., 2019; Lim et al., 2015; Lim & Lee,

2016; Nakashima et al., 2015; Park et al., 2018; Schick et al., 2007). This work suggests that the Ptenlfl/fl; CamKIIα-Cre mouse is useful for the study of epilepsy. This model and our findings in Chapter 4 may be relevant for a subset of individuals with FCD if there is a driving mutation in

PTEN. Additionally, given the knowledge that p110β overexpression occurs in other neurodevelopmental disorders, it is plausible that there may be a supplementary subset of FCD patients which may benefit from inhibition of p110β due to overexpression. This idea could be further elucidated by measuring p110β levels in other disorders which contribute to FCD.

As mentioned multiple times, one interesting finding of previous studies assessing mutations in PTEN in both cancer and autism is that they vary in each disorder (Fricano-Kugler et al., 2018; Spinelli et al., 2015). Work assessing these mutations concluded the mutation

224 affects the cellular phenotypes and thus the resulting disorder, as discussed in the introduction to this work. Given this knowledge, it is plausible that, although we reported favorable outcomes in our PTEN-deficient mouse models, the therapies associated with cancer may not yield a favorable response in autism associated with PTEN mutations. With the variance from the mutations, proteins, and disorders in mouse models compared to humans, it is difficult to predict this without the use of a clinical trial which test the effects of p110β inhibition.

Concluding Remarks

The implications of PTEN deficiency on a multitude of disorders is still an expanding field. From its original association to cancer, the list of PTEN-linked disorders has continuously developed. I have explored PTEN and some of the therapeutic approaches as well as the implications of these approaches in both brain tumors and autism.

Given the previously mentioned concern about the gap between mouse models and humans, it is yet to be determined if some of the favorable outcomes reported here will translate to improved treatments for both cancer and autism.

For the next approximate year, I intend to move the p110β project (Chapter 4) forward in hopes of continuing to gain a better understanding of the outcomes of p110β inhibition in autism and seizure disorders associated with PTEN deficiency.

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Tables

Pathway Or Cellular Genes Involved Process Dysregulated PI3K BCL2, CDKN1B, HRAS, KIT, MAPK1, MET, MTOR, PTEN, TSC1, TSC2, YWHAE mTOR BRAF, HRAS, MAPK1 Ras GRIN2A, HRAS, KIT, MAPK1, MET, NF1, PTPN11 MAPK BRAF, CACNA1D, HRAS, MAPK1, NF1, NTRK1 Chromatin CHD8, CHD7, CHD2, Remodeling ARID1B, ATRX. ATRX Genome ERCC6, RAD54, Maintenance HTLF, SHPRH, RAD16 Transcription Factors ADNP, PAX5, FOXP1, TCF7L2, TBLXR1

Table 1: Shared pathways and cellular processes dysregulated in cancer and autism.

Beyond the PI3K pathway, additional pathways associated with both cancer and autism include mTOR, Ras, and MAPK. Note the literature separated out mTOR as some of these regulations are separate from PI3K. Cellular processes which are found to be dysregulated in both cancer and autism include chromatin remodeling, genome maintenance and transcription factors.

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Group N Therapy 1 Therapy 2

1 10 PX-866 once daily by mouth x7 days, Rapamycin Saline ID x1

once daily IP x7 days

2 10 PX-866 vehicle once daily by mouth x7 days, Dendritic cell ID x1

Rapamycin vehicle once daily IP x7 days

3 10 PX-866 once daily by mouth x7 days, Rapamycin Dendritic cell ID x1

once daily IP x7 days

4 10 PX-866 vehicle once daily by mouth x7 days, Saline ID x1

Rapamycin vehicle once daily IP x7 days

Table 2: Hypothetical experimental design for mouse study which targets both differentiated and stem cells. PX-866 and rapamycin are used for the purpose of inhibiting

PI3K and dendritic cell is used for the purpose of targeting the undifferentiated (stem or stem- like) population. N=number per group. IP=intraperitoneal. ID=intradermal.

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Group N Treatment

1 10 BKM120 once daily (by mouth) for one week

2 10 temozolomide once daily (by mouth) for one week

BKM120 once daily (by mouth) for one week; temozolomide once daily (by 3 10 mouth) for one week

BKM120 vehicle once daily (by mouth) for one week; temozolmide vehicle 4 10 once daily by mouth for one week

Table 3: Hypothetical experimental design for patient-derived xenograft study. BKM120 is the pan PI3K inhibitor previously utilized in our pHGG work. Temozolmide represents a standard therapy used in humans. N=number per group.

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Compound Clinical Signs Examples Clinical Findings Antidepressants (SSRIs irritability, repetitive Fluoxetine, fluoxetine and and SNRIs behaviors, and venlafaxine clomipramine compulsions reduces repetitive behaviors compared to controls, a relatively large randomized controlled trial (RCT) of citalopram in children failed to show any benefit Stimulants ADHD Methylphenidate ASD patients show less response as compared to children without ASD Alpha-2 agonists Hyperactivity and Clonidine, guanfacine shows impulsivity guanfacine significant reduction in hyperactivity as reported by parents Atomoxetine Hyperactivity Atomoxetine ASD patients show less response as compared to children without ASD Atypical antipsychotics Aggression, Risperidone and Reduce many tantrums and self- Aripiprazole effects but include

injury severe clinical signs including sedation and weight gain Oxytocin Social impairments oxytocin Reduced repetitive and repetitive behaviors in some behavior patients, no other clinical changes GABA-B agonist Social impairments arbaclofen improvements in irritability and social withdrawal scales, obsessive- compulsive symptoms, and social responsiveness NMDA antagonist Communication memantine Reported positive deficiency effects but many severe side effects N-Acetylcysteine Irritability NAC No substantial evidence in humans

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Table 4: Current therapeutic approaches used for the treatment of autism symptoms.

Compound classifications are grouped to address the specific clinical signs they are used to treat. SSRI= selective serotonin reuptake inhibitor, SNRI= serotonin-norepinephrine reuptake

Inhibitor, ADHD= attention deficit hyperactivity disorder, ASD= autism spectrum disorders,

NMDA= N-Methyl- d-aspartate, NAC= N-acetyl cysteine

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