Tamoxifen Therapy in a Murine Model of Myotubular Myopathy

by

Nika Maani

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics

University of Toronto

© Copyright by Nika Maani 2018

ii

Tamoxifen Therapy in a Murine Model of Myotubular Myopathy Nika Maani

Department of Molecular Genetics University of Toronto

2018 Abstract

X-linked myotubular myopathy (XLMTM), also known as myotubular myopathy (MTM), is a

fatal pediatric congenital myopathy caused by loss-of-function mutations in MTM1 that is without

existing therapy. MTM1 is a phosphoinositide 3-phosphatase that antagonizes class II and III

phosphatidylinositol 3-kinases (PI3Ks) to modulate levels of endosome-specific

phosphoinositides. MTM1 regulates endosomal trafficking, and proper formation of the skeletal

muscle triad. Using a murine model of MTM, our lab has identified tamoxifen as a novel

therapeutic candidate. Using several in vitro and in vivo studies and RNA sequencing, I

demonstrate the aforementioned effects of tamoxifen to be mediated primarily through estrogen

receptor signaling, and the post-transcriptional regulation of dynamin-2 (DNM2), a known MTM1

modifier. The preclinical evidence presented herein identifies and uncovers the FDA approved

drug tamoxifen as the first small molecule therapeutic with pre-clinical efficiency and clinical

translatability for use in MTM patients.

iii

Dedication

To my loving parents and dedicated husband

For this journey would never have come to fruition without you.

To my parents, who have given me unwavering and unconditional material and spiritual

support throughout every step of my life and academic pursuits. Your never-ending sacrifice of

resources, time and emotion can never be matched or replicated. Thank you for teaching me how

to overcome the setbacks and embrace the triumphs that accompany a life of sacrifice and service

with an unimaginable level of elegance and dedication. Thank you for making the decision that

would forever change your entire lives, leaving behind everything you knew and loved, so that I

could live the life you couldn’t.

To my husband, the biggest source of light in my life. Thank you for always pushing me

to overcome the barriers and limits I had set in the past, and to see myself in ways I never knew I

could. To me, you are the embodiment of true love and sacrifice. Thank you for continually

teaching me how to always put the needs of others above your own with an unparalleled sense of

steadfastness. Thank you for your never-ending faith, and for going through all my highs and lows

as if they were your own.

I could only hope that I have given you as much love and devotion as you have given and

continue to give me.

iv

Acknowledgments

The completion of this work would not have been possible without the unconditional support of

my supervisor Dr. James Dowling. One of the greatest privileges of working under Dr. Dowling

is the ceaseless opportunity to witness his resolute commitment towards improving the lives of

children and families afflicted with neuromuscular disorders worldwide, as a renowned physician

and respected scientist, with a level of humanity and reverence that is singular in nature.

Thank you for not only showing me what it looks like to dedicate your life towards the service of

others, but for also pushing me to undertake the herculean efforts required to do so. Thank you for

seeing potential and intellect in me when I never did and thank you for building me a space that

allowed me to fully explore how I wish to dedicate and build my future in Canadian healthcare.

I would also like to thank the members of my supervisory committee Dr. Ronald Cohn and Dr.

Lucy Osborne for always giving me excellent professional and academic guidance on my path to

becoming a better graduate student, critical thinker and contributor towards scientific

advancement.

In addition to the unmatched mentorship and support I’ve received from my supervisor and

committee, I have been extremely fortunate to receive further degrees of enrichment from all my

lab mates and collaborators at the Hospital for Sick Children and the University of Toronto. In

particular, I would like to thank Jonathan Volpatti, Hernan Gonorazky, Arun Ramani, Lindsay

Smith and Nesrin Sabha for their true friendship, accompaniment and unconditional willingness

to share their expertise as I worked towards the completion of this project.

Lastly, my time at SickKids would not have been complete without the former, current and new

members of the Dowling lab, all of whom I regard as my Torontonian family.

v

Table of Contents

Contents Dedication ................................................................................................................................. iii Acknowledgments ..................................................................................................................... iv Table of Contents ........................................................................................................................ v Chapter 1 Introduction ................................................................................................................ 1 1 X-linked Myotubular Myopathy and Other Congenital Myopathies ........................................ 1 2 Function, Structure, and Localization of MTM1 and DNM2 ................................................... 5 3 Genetic Landscape of MTM1 and DNM2 ............................................................................... 8 4 Known Pathogenic Mechanisms in XLMTM and ADNCM .................................................... 9 5 Therapeutic Landscape of XLMTM ..................................................................................... 13 6 Pathophysiological Characterization of Mtm1 KO Mice and Mtm1 KO Mice Treated with

Tamoxifen ............................................................................................................................ 15 7 Estrogen Signalling as a Modulator of Skeletal Muscle Structure and Function .................... 17 8 Proteostasis as a Mechanism of Skeletal Muscle Maintenance .............................................. 18 9 Summary .............................................................................................................................. 20 Chapter 2 In-Vivo Analyses of the Expression and Localization of Estrogen Receptors in

Mtm1 KO mice and Tamoxifen-treated Mtm1 KO mice ........................................................ 23 10 Expression of Estrogen Receptors ........................................................................................ 23 11 Localization of Estrogen Receptor Alpha (ERa) .................................................................. 29 12 Analysis of Whole-RNA sequencing in Mtm1 KO and Tamoxifen-treated Mtm1 KO

Murine Models. .................................................................................................................... 31 13 Transcriptional Landscape of Mtm1 KO mice ....................................................................... 33 14 Effect of Tamoxifen on the Transcriptional Landscape of Mtm1 KO mice ............................ 40 Chapter 3 In-Vivo Analyses of the Expression of PIK3C2B and DNM2 in Mtm1 KO mice

and Tamoxifen-treated Mtm1 KO mice ................................................................................. 45 15 Expression and Activity of PIK3C2B ................................................................................... 45 16 Expression and Modulation of DNM2 .................................................................................. 48 17 Protein signatures in Mtm1 KO Mice Following Treatment with 17b-estradiol and

Fulvestrant ........................................................................................................................... 52 Chapter 4 In-vitro Analyses in Murine and Human Cellular Models to Identify Tamoxifen’s

Therapeutic Mechanism of Action in XLMTM..................................................................... 57 18 Estrogen Receptor Dependence of Tamoxifen in XLCNM ................................................... 59 19 Investigating the mechanism of action of Tamoxifen ............................................................ 68 Chapter 5 Discussion ................................................................................................................ 74 Chapter 6 Future Directions ...................................................................................................... 81 20 Aim 1: Further Explore the Role of Tamoxifen as a Post-Translational Modulator of

DNM2. ................................................................................................................................. 82 References ................................................................................................................................ 86

vi

Appendix I: Materials and Methods .......................................................................................... 92 Appendix II: Supplementary Tables .......................................................................................... 96

1

Chapter 1 Introduction

1 X-linked Myotubular Myopathy and Other Congenital Myopathies

Congenital myopathies encompass a genetically and clinically heterogeneous group of

neuromuscular conditions characterized by distinctive structural and functional abnormalities in

skeletal muscle1,2. Despite sharing a number of common clinical features such as: childhood-onset

hypotonia, progressive muscular weakness and atrophy3, the conditions grouped under this

diagnostic umbrella have been further subdivided into morphologically distinct groups.

Centronuclear myopathies (CNMs) encompass a group of rare, and genetically diverse congenital

myopathies that have been aptly named by way of their presentation of skeletal myofibers with

significantly prevalent centralized nuclei on muscle biopsy2,3. Since their first description in 1966

by Spiro et al., CNMs have been further classified into three subgroups (X-linked, autosomal

dominant, and autosomal recessive) as a consequence of distinct genetic and clinical features that

underlie their spectrum of clinical severity that ranges from neonatal lethal to adolescent/adult

onset and slowly progressive. Most importantly, all CNMs are associated with severe and lifelong

disabilities and are uniformly without therapies at present1–3.

To date, mutations in six different genes have been associated with CNMs; with mutations in

myotubularin (MTM1), being the most commonly occurring and extensively studied4–7. The other

four classical CNM genes include dynamin-2 (DNM2)8–10, amphyphysin 2 (BIN1)11–13, ryanodine

receptor 1 (RYR1), titin (TTN) and SPEG1,14. Whilst being less common than MTM1, mutations in

the latter genes have been associated with the rarer and oftentimes milder autosomal dominant and

autosomal recessive forms of the disease, respectively1–3. To date, functional abnormalities in

MTM1, DNM2 and BIN1 have been repeatedly implicated in CNM pathology and have been

hypothesized to play important molecular roles at the transverse (T) tubule: a muscular

substructure specialized for calcium handling8,9,12,15–17. Moreover, these genes all possess functions

in membrane remodelling, endocytosis and vesicular trafficking9,17–19. However, the molecular

mechanisms underlying the clinical and pathological similarities between MTM1, DNM2 and

BIN1-related CNMs to those caused by mutations in RYR1 and TTN are unclear19. Caused by in

utero loss of function mutations in MTM1, X-linked MTM (XLMTM) (commonly known as

myotubular myopathy (MTM)), has an estimated prevalence of 1-9 in 100,000 male births, and is

2

also the most severe form of CNM3,4,6 . Patients often present with profound muscular weakness

or hypotonia at birth, accompanied by the acquisition of progressive and severe disabilities

(including wheelchair and ventilator dependence). Opthalmoparesis, cryptorchidism and the

elongation of facial features are additional distinguishing abnormalities2,20. Although the majority

of patients do not survive past infancy as a consequence of respiratory failure, those surviving into

late childhood or early adolescence, often require invasive ambulatory, respiratory and

gastrointestinal support. Of those surviving few, many will die before reaching adulthood14.

Patients affected with autosomal dominant (ADCNM) and autosomal recessive (ARCNM) forms

of CNM also present with moderate to severe muscle weakness and delayed motor milestones

along with facial and/or ocular abnormalities2,19. Oftentimes male patients affected with either X-

linked or autosomal forms of the disease may be indistinguishable by clinical observation and

muscle biopsy alone and require confirmatory gene testing to reach a conclusive diagnosis2.

Moreover, by virtue of such diagnostic advances in whole-genome, -exome and RNA sequencing

technologies, additional neonatal and more severe forms of ADCNM have been identified as a

result of their association with de novo DNM2 mutations2,10,21. Occasionally however, autosomal

CNMs can be distinguished from MTM by virtue of their additional incidence in females, most-

often later period of onset, and supplementary clinical manifestations such as ptosis, scoliosis and

limb-girdle patterns of muscle weakness2,19,21.

Both autosomal and X-linked forms of CNM present with similar features on muscle biopsy; the

hallmark identifier being the presence of centralized nuclei in over 25% of muscle fibers.

Furthermore, the majority of affected fibers are significantly smaller, rounder and resemble an

immature muscular phenotype2,14,19. The predominance of hypotrophic type 1 muscle fibers

however, is a histological feature of MTM, whilst the appearance of radial sarcoplasmic strands

observed by oxidative enzyme staining represent a histopathological feature of ADCNM2. Despite

oftentimes complicating genotype-phenotype correlations amongst CNMs, the extensive clinical

and histological overlap does raise the interesting question of pathological interrelatedness. At

present, the specific reason(s) as to why mutations in different genes result in similar muscle-

specific abnormalities remains to be clearly defined. Furthermore, whether the gene-products of

MTM1, DNM2 and BIN1 all function within a single pathway or co-localize within a specific

muscular region to exert their interdependent molecular function(s) remains an open and

extensively investigated question.

3

Figure 1. Speculated molecular and functional links between BIN1, MTM1 and DNM2 in skeletal muscle. T-tubules are muscle-specific organelles that are critical for calcium signalling and excitation-contraction coupling. They also link the sarcoplasmic reticulum and the cytoplasm (sarcoplasm) of skeletal muscle at the triad by way of molecular interactions between dihydropyridine receptors (DHPR) and ryanodine receptors (RYR1). MTM1 (red circles) functions to regulate vesicular trafficking, ensuring the proper transport of muscle-specific proteins. BIN1 is involved in membrane remodelling and membrane tubulation, in concert with Caveolin-3 (Cav3)/Caveloae at invaginating regions of the plasma membrane. DNM2 is speculated to function in parallel to BIN1 as a regulator of membrane fission, to modulate T-tubule formation and maintenance, however the exact molecular function and/or role of DNM2 at the T-tubule is poorly understood.

To exert its specialized function, skeletal muscle requires specialized structures known as the triad

and neuromuscular junction (NMJ)22. Both the triad and NMJ function as key regulators of EC

coupling, the process whereby nerve signals are translated into contractile force via the

intracellular release of Ca2+.22 Briefly, following motor neuron-induced excitation at the NMJ, a

wave of depolarization is propagated along the cellular membrane and through a specialized

sarcolemmal invagination known as the Transverse (T)-tubule. Surrounded on each end by

terminal ends of the sarcoplasmic reticulum (SR), this T-tubule-SR complex is known as the triad

and is responsible for translating membrane depolarization into muscle contraction by promoting

the mechanical release of Ca2+ into the cell19,22. Intriguingly, MTM1, DNM2 and BIN1 have all been

directly or indirectly implicated in the formation and/or maintenance of the triad8,11,12,17,18,22 (Figure

2). Furthermore, one pathologic mechanism that unifies autosomal and X-linked forms of CNM is

in fact the disruption of myofiber architecture and specifically, structural disorganization of the

triad and NMJ2,14. Undoubtedly, structural changes of the triad hold significant functional

4

relevance in MTM muscle and have been shown to underlie the clinical manifestations of muscle

weakness and impaired muscle contraction seen in patients2,14,19. It is therefore possible that

muscle-specific abnormalities arise in CNMs by virtue of the dysfunctional interplay of these

genes at the triad.

Despite the strong pathological and clinical classifications discussed above, the long-term

prognosis of MTM remains very poor, with a disease burden that is potentiated by the lack of an

effective therapy or cure. By virtue of its high rate of infantile mortality, MTM is regarded as the

most severe and devastating form of CNM2,14,19. Predicting the clinical outcomes of MTM patients

is especially difficult without a comprehensive understanding of the natural history of the disease

and the discovery of well-defined prognostic indicators. In light of this, a prospective, non-

interventional clinical assessment study (INCEPTUS; NCT02704273) is currently underway to

characterize the disease course, identify therapeutic windows and establish outcome measures that

can be used to assess the effectiveness of novel therapies. In a similar vein, three natural history

studies20,23,24 have been conducted in the past and provided some insight into actionable and/or

measurable disease outcomes in MTM. Earlier studies from 199923 and 200224 showed

significantly high rates of mortality in MTM patients and ascertained that survival beyond one year

of life is often a reflection of the extensive provision of medical and respiratory support that is

necessary to fight against severe non-muscle morbidities23,24. Furthermore, one group determined

that gene mutation analyses in MTM are unreliable for generating genotype-phenotype

correlations24. In the most recent study however, Amburgey et al., showed an increase in patient

survival to an average of 6 years and 10 months (83% of patients died before age 9), despite similar

disease severities and morbidities as those observed in previous studies20. Amburgey and

colleagues identified respiratory status as the most promising disease outcome measure, given that

use of motor scales, frequency of hospital visits and overall survival was found to be limited as a

prognostic indicator20. Despite these advances however, no proven therapy or disease modifying

therapy exists. In light of its devastating severity, many groups have set the stage for a novel

therapeutic era by exploring whether the phenotypic overlaps amongst CNMs are a consequence

of a unifying genetic pathomechanism. Indeed, DNM2 has recently been identified as a novel

genetic modifier of MTM125,26, and therefore not only sets the precedent for a novel therapeutic

approach, but also helps to further our understanding of MTM pathology. Consequently, the

information presented hereafter will focus preferentially on the structure, function and molecular

interrelation of MTM1 and DNM2 within the context of MTM.

5

2 Function, Structure, and Localization of MTM1 and DNM2

MTM1 protein, also known as myotubularin, is a lipid phosphatase that dephosphorylates

specialized membrane lipids known as phosphoinositides (PIPs) at the 3’ position. Specifically,

MTM1 antagonizes the action of PIK3C2B, a phosphatidylinositol 3-phosphate (PI3P) kinase, to

convert PI3P to PIP and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) to PI5P7 (Figure 2).

PIPs are lipid second messengers that are important mediators of cellular signalling and membrane

transport. Phosphatidylinositol 4-phosphates (PI(4)P and PI(4,5)P2) are enriched at plasma

membranes, secretory organelles and lysosomes, whilst PI(3)P and PI(3,5)P2 function as regulators

of membrane trafficking, intracellular sorting and organelle maturation at the endosome7,27,28. In

this way, MTM1 functions to regulate the proper sorting and recycling of endosomal proteins by

maintaining balanced levels of phosphorylated and dephosphorylated PIPs on membranes of the

endolysosomal system28. The significance of this regulation is highlighted by the fact that

mutations in many PIP-modifying enzymes, including MTM1, have been implicated in several

neuromuscular and multi-factorial diseases19. Interestingly however, MTM1 has yet to be detected

at the nucleus or triad. A study conducted in C2C12 mytoubes and myoblasts found MTM1 to be

localized preferentially at the cytoplasm and plasma membrane29, whereas two studies in MTM

fibroblasts and skeletal muscle, found MTM1 at sorting and recycling endosomes27 as well as

regions of the sarcoplasmic reticulum18, respectively. Not surprisingly, an important question in

the field remains: how does the in vitro function of MTM1 as a regulator of endosomal dynamics

and PIP metabolism associate with muscle-specific and in vivo pathologies of triad dysregulation

and nuclear centralization? There is a growing hypothesis that defects in the exit of protein cargoes

from endosomes may underlie the accumulation or sequestering of proteins required for muscular

maintenance and overall function. In support of this, Ketel et al demonstrated that MTM patient

fibroblasts harbour abnormalities in the endosomal recycling of b1-integrin and transferrin

receptor (TfR)27. Furthermore, in a more recent study, they identified PI4K2a and Sec6 as

interactors of MTM1 by immunoprecipitation27. PI4K2a is a phosphatidylinositol 4-kinase that

phosphorylates PIP to PI(4)P30, and Sec6 is a component of the exocyst complex that enables

fusion of exocytic vesicles to the plasma membrane31. Briefly, MTM1 and PI4K2a were shown to

initiate the conversion of PI(3)P to PI(4)P at the recycling endosome to assign the correct

membrane identity required for its proper fusion with the plasma membrane27. Whether MTM

functions in a similar manner to promote the exocytic regulation of muscle-specific proteins from

6

endosomal compartments to their correct subcellular locations however, remains to be determined.

Moreover, whether the accumulation of exocytosis-deficient endosomes is a primary driver of the

muscle-specific pathologies observed in patient and murine models of MTM requires further

investigation.

Figure 3. MTM1 is a lipid phosphatase that negatively regulates phosphatidylinositol 3-phosphate (PI3P), antagonizing the function of class II and class III phosphoinositide 3-kinases (PI3K). MTM1 is involved in regulating endolysosomal trafficking, membrane remodeling, and autophagy. MTM1 is a member of a family of 14 highly conserved active and dead phosphatases. A notable

characteristic of the myotubularin family is homo- and hetero-dimerization of active phosphatases

with their catalytically inactive homologs (i.e. MTM1/MTMR12), through the PH-GRAM and CC

protein domains, to promote the localization, stability and allosteric activation of MTMs at PIP-

enriched membranes7. Lastly, PTP (catalytic) and Rac-1 induced localization (RID) domains are

both subject to pathogenic mutations in MTM patients7,19. Interestingly, each domain is responsible

for promoting phosphatase activity, or targeting MTM1 to specific membrane regions,

respectively7. It is therefore likely that further investigation into the biological consequences of

specific mutations in these domains will shed insight into how dysregulation of the endocytic

machinery may promote development of muscle-specific pathologies.

In a similar vein, DNM2 is one of three members of the dynamin superfamily of GTPases that

shape and remodel membranes throughout diverse cellular processes. Unlike DNM1 which is

mainly expressed in the brain, and DNM3 in the brain and testes, DNM2 is expressed and functions

more ubiquitously10,19. The most extensively studied function of DNM2 however, is its role as a

key regulator of clathrin-mediated endocytosis (CME)8. In C2C12 cells and fibroblasts, DNM2 is

localized to the cytoplasm and clathrin-coated pits at the plasma membrane8. CME is the principal

mechanism by which cells internalize protein cargo (i.e. transferrin, growth factors, etc.) that are

bound to specific membrane receptors, and therefore integral to proper synaptic vesicle recycling32,

cell signaling and growth33. CME is a multi-step process mediated by a modular and transient

7

complex of over fifty different endocytic proteins recruited to plasma membrane regions enriched

for specific PIPs (i.e. PI(4,5)P2)11,17. The discovery of PIP binding domains within certain endocytic

proteins suggests that PIP regulation may play an important role in the assembly and targeting of

endocytic protein complexes. The PH domain of DNM2 is speculated to bind directly to PI(4,5)P2,

mediating its recruitment to endocytic membranes in preparation for membrane scission34. Briefly,

cytosolic tetramers of DNM2 have been shown to oligomerize at the neck of clathrin-coated

vesicles following recruitment to PIP-rich membrane regions34. Subsequent constriction and

scission of the plasma membrane however, is speculated to be driven by mechanical forces

transmitted throughout the protein following GTP hydrolysis33,34. Consequently, the GTP

hydrolysis-dependent membrane remodeling activity of DNM2 is understood to depend on proper

oligomerization34. DNM2 is composed of five domains: an N-terminal GTPase domain, the stalk

and bundle signalling element (BSE) domains regulating dynamin self-assembly, a pleckstrin

homology (PH) domain responsible for binding membrane-bound lipids (i.e. PI(4,5)P2)), and a C-

terminal proline-rich domain (PRD) that mediates interactions with -BAR and -SH3 domain-

containing scaffolding proteins12,34. Despite extensive studies investigating the molecular

architecture of DNM2, most have failed to clarify the exact mechanical mechanism(s) that govern

dynamin assembly, function and regulation. Recently however, a biochemical study of the crystal

structure of DNM2 by Reubold et al suggests that DNM2 oligomerization occurs following release

of an intramolecular auto inhibitory mechanism34. Intriguingly, this group hypothesized that

mutations in domains involved in oligomerization and auto inhibition (i.e. PH and BSE domains)

may have direct implications for DNM2 function in membrane constriction and scission. Not

surprisingly, they speculate that defects in this mechanism contribute to the development of CNM

pathophysiology34. A growing body of evidence suggests that DNM2 may also play a role in

membrane tubulation by virtue of its speculated interaction with BIN1, another CNM gene and

endocytic protein localized to PI(4,5)P2—enriched membranes11–13. In light of its well-established

role as a sensor and inducer of membrane curvature, recent studies suggest that BIN1 mediates T-

tubule biogenesis and is capable of recruiting DNM2 to endocytic sites via its C-terminal SH3

domain11,17. Indeed, in the absence of MTM1, BIN1 is no longer found at the t-tubule; suggesting

that defective membrane tubulation may underlie the structural disorganization of the triad in

myopathic muscle25. Although DNM2 partially co-localizes with BIN1 labelled tubular structures

in C2C12 myotubes, and caveolin-3 labelled t-tubule-adjacent structures8, whether DNM2 and

BIN1 directly interact is not clear; nor is it completely understood whether either protein functions

to regulate the other within skeletal muscle. This is in contrast to MTM1, which co-localizes with

8

BIN1 at the T-tubule and when bound, enhances BIN1-mediated membrane tubulation in vitro35.

Whether the involvement of MTM1 and/or speculated role for DNM2 in T-tubule formation

contributes towards triad dysregulation in murine models of CNM however, merits further

investigation.

Further evidence has implicated DNM2 in actin-dependent trafficking, cytoskeletal assembly and

centrosome cohesion, all of which are of potential relevance to mechanisms underlying abnormal

nuclear positioning in CNM36,37. This is supported by a recent case study in which the subcellular

localization of DNM2 was disrupted in an ADCNM patient with a novel mutation in the C-terminal

region of the PH domain15. In skeletal muscle biopsies, DNM2 localized at the surface of

centralized nuclei as puncta aggregates; this was in contrast to control biopsies in which DNM2

localized to the periphery of muscle fibers. Not surprisingly, this was accompanied by profound

changes in muscle fiber morphology15. Taken together, this data supports the idea that MTM1 and

DNM2 play important roles in intracellular trafficking and membrane remodelling. In this way,

they both function to promote protein sorting into correct intracellular compartments. However,

the relationship between the molecular functions of MTM1 and DNM2 with that of the structural

and functional abnormalities in myopathic muscle is unknown.

3 Genetic Landscape of MTM1 and DNM2 MTM1 is located on the X chromosome (Xq28.1) and is as such, causative of an X-linked recessive

disorder, with a penetrance of 100% in all males that carry a pathogenic variant6,14,19. Upwards of

300 MTM1 mutations have been reported in the literature, with 529 variants submitted to the

Leiden Open-source Variation Database (LOVD)38. Mutations are distributed indiscriminately

along the entire gene, with no preferential clustering to loci encoding known functional domains.

Furthermore, the majority of mutations have been found to be exonic, with a minor subset found

within introns and intron-exon boundaries6,19. Consequently, functional relevance and clear

genotype-phenotype correlations for MTM1 mutations have yet to be established. Unsurprisingly

however, most truncating mutations (deletions or nonsense) and missense mutations of the PTP

domain have been associated with more severe phenotypes, whilst non-truncating mutations and

those outside the PTP domain have been identified in less affected individuals6,19,20,24.

DNM2 maps to chromosome 19 (19p13.2-p12) and contains 22 exons. Mutations in DNM2 are

heterozygous and dominant; pathogenic mutations are often missense or in-frame indels, with the

9

majority clustering to the PH domain and interface of PH and stalk domains2,19. At present, 60

variants have been reported in the LOVD, six of which (R465W, S619L, A618T, E386K, V625

and R369W) have been extensively studied and characterized38. The recurrent p.R465W mutation

is the most common, and accounts for approximately 25% of affected families, whereas the

p.E368K, p.R369W mutations along with those in residues 618 and 619 are found in approximately

20%, 10% and 15% of families, respectively19. ADCNM affects both males and females with an

overall decreased disease severity than that of MTM. Despite this however, more severe and

neonatal onset forms of ADCNM have been associated with heterozygous de novo mutations

within the PH domain2,14,19. Intriguingly, Reubold et al., have recently proposed a novel structural

mechanism of DNM2 assembly and regulation through which the effects of disease-related

mutations may be explained. They propose an intramolecular mechanism of auto-inhibition in

which the PH domain remains bound to the stalk to prevent unnecessary oligomerization.

Following the recruitment of DNM2 dimers to pre-endocytic sites by accessory and scaffolding

proteins, this inhibitory molecular “switch” is turned “off” by virtue of PH domain binding to

membrane-specific PIPs (i.e. PI(4,5)P2); thus, modulating the stepwise assembly of tetrameric

helices around invaginating membranes34. Interestingly, when expressed in mammalian cells,

DNM2 lacking the PH domain preferentially forms cytosolic aggregates, and fails to localize to

clathrin-coated membranes and modulate transferrin uptake34. Reubold and colleagues propose

that CNM-causing mutations within, or at the interface of the PH and stalk domain interfere with

the modulation of this auto-inhibitory mechanism, and downstream GTPase activity of DNM2. By

virtue of its location it is speculated that the R465W mutation disrupts the intramolecular

interactions required for DNM2 assembly and regulation, and thus promotes excessive DNM2

oligomerization834. Furthermore, the fact that Liu et al speculate ADCNM-associated mutations in

Dnm2 to be “gain-of-function” or hypermorphic, further supports the prevalent hypothesis that

DNM2 over activity is a primary pathological mechanism in ADCNM. Although not immediately

obvious, these findings provide important insight into pathomechanisms of MTM, a concept which

will be further elaborated in subsequent sections.

4 Known Pathogenic Mechanisms in XLMTM and ADNCM As a consequence of the extensive work carried out in well-established mammalian models of

MTM, several pathologic mechanisms have been proposed to explain why several structural and

physiologic abnormalities of the disease may arise. At present, three murine models that faithfully

recapitulate the pathology of the human disease have been developed39–41. Although triad and NMJ

10

dysfunction represent structural abnormalities of the disease, the molecular reason(s) for which

they arise is unclear14. Notably, all three murine models share hallmark pathological features of

accumulated PI(3)P, abundant centralized nuclei, decreased fiber size, predominance of type I

fibers, and structural dysregulation of the triad39–41. Not surprisingly, all three models exhibit

severely reduced muscle strength, with two of the three recapitulating more severe and mild forms

of MTM39,40. Notwithstanding their pathogenic similarities, the use of each model has provided

important insights into different pathomechanisms that may underlie the hallmark structural and

molecular abnormalities of the disease.

The first and most extensively studied murine model was described and developed in 2002 by Buj-

Bello et al. and is commonly referred to as the 129pas strain. Buj-Bello and colleagues used Cre-

Lox mediated homologous recombination to knock-out exon 4 in Mtm1, generating a truncating

mutation that caused the complete loss of MTM139. Not surprisingly Mtm1 KO mice have greatly

reduced lifespan (median survival = 35 days) compared to wild-type littermates, and faithfully

represent severe disease phenotypes. In addition to its extensive characterization in the literature,

the Mtm1 KO (MTM) mouse model was most notably used by our lab and Cowling et al and has

provided important insights into two attractive pathogenic mechanisms of the disease25,26,42. By

virtue of myotubularin’s role as a PI(3)P phosphatase, it is possible that many structural

abnormalities in the muscle arise as a consequence of PI(3)P accumulation following the loss of

MTM11,4. In support of this, our lab showed that genetic depletion of Pik3c2b in Mtm1 KO mice

was sufficient to reduce PI(3)P levels, and rescue the lethality of the Mtm1 KO mouse model.

Importantly, our findings introduced Pik3c2b as one of the first genetic modifiers of MTM and

demonstrated that PIK3C2B inhibition represents a novel and excellent therapeutic strategy for the

disease42. Moreover, the therapeutic benefit of reducing PI(3)P levels in the Mtm1 KO mouse

demonstrates that nuances in PIP metabolism may contribute towards development of hallmark

muscular pathologies in MTM. This hypothesis is in line with findings from Amoasii and

colleagues, where both PI(3)P and MTM1 were localized to sarcoplasmic cisternae in skeletal

muscle of Mtm1 KO mice18. Moreover, when the phosphatase activity of MTM1 was altered in

skeletal muscle, they observed abnormal remodeling of the sarcoplasmic reticulum18,43. Coupled

with the understanding that MTM1 binds to, and acts in conjunction with BIN1 to promote

membrane tubulation in vivo16, it is possible that MTM1 activity and the consequent regulation of

PI(3)P plays a key role in maintaining the structural integrity of the sarcoplasmic reticulum at the

triad. Although triad abnormalities are among the first changes identified in MTM and likely

11

explain the profound weakness seen in MTM patients2,14, the association between MTM1, alerted

PIP metabolism and triad dysfunction has yet to be translated into corresponding therapies for

MTM patients and requires further investigation. Clarifying the pathogenic consequences of PI(3)P

accumulation in MTM will shed insight into MTM pathogenesis and the etiology of muscle-

specific abnormalities (i.e triad dysregulation and nuclear centralization). This knowledge can then

be applied towards understanding the pathology of other neuromuscular diseases and CNMs that

arise as a consequence of altered PIP metabolism.

Another discovery of relevance to MTM pathogenesis comes from Cowling et al. who recently

identified Dnm2 as another novel genetic modifier of Mtm125,26. Using the Mtm1 KO mouse model,

Cowling and colleagues demonstrated that DNM2 protein expression was significantly elevated in

human MTM fibroblasts and skeletal muscle of the Mtm1 KO mouse. Taking advantage of the fact

that heterozygous Dnm2 -/+ mice are phenotypically normal, Cowling and colleagues reduced

DNM2 protein levels by approximately half in Mtm1 KO mice by generating Mtm1 KO mice that

were heterozygous for Dnm2 (Dnm2-/+Mtm1-/-). Remarkably, Dnm2 reduction was sufficient to

rescue the survival, muscle function and triad structure of Mtm1 KO mice9,25,26. Following this

discovery, Tasfaout et al successfully downregulated DNM2 in Mtm1 KO mice using anti-sense

oligonucleotides (ASOs) and intramuscular injection of AAV-shRNA against Dnm244,45(short

hairpin RNA sequences against DNM2 mRNA); both methods successfully rescued MTM

pathology and represent clinically relevant strategies of DNM2 reduction. In wild-type murine

muscle, overexpression of CNM-associated Dnm2 mutations (i.e. R465W) has been shown to

induce nuclear centralization, muscular atrophy and triad dysregulation. Similarly, similar

pathologic features are observed when wild-type Dnm2 is overexpressed in cellular and murine

models8,9. These models along with established murine models of ADCNM recapitulate the

pathological abnormalities often observed in ADCNM patients, and elegantly demonstrate that

DNM2 overexpression is an independent inducer of pathogenicity. This is consistent with the

hypothesis that DNM2 hyperactivity is a likely pathogenic mechanism in both diseases and

suggests that the mechanism(s) underlying DNM2 dysregulation in ADCNM and MTM are

similar8. It is therefore possible that DNM2 overexpression drives the development of muscular

abnormalities observed in both diseases. Given that DNM2 has been implicated in CME, the

regulation of actin and cytoskeletal dynamics, and indirectly associated with the biogenesis of the

T-tubule, it is unclear as to which function of DNM2 contributes to MTM pathogenesis. This is

further evidenced by the lack of any interaction between MTM1 and DNM2, to date. What is clear

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from these findings however, is that DNM2 and MTM1 function together in a common, but

incompletely understood pathway to maintain specialized structures in skeletal muscle. This is

evident given the significant phenotypic overlap amongst all CNMs2. Most importantly however,

this indicates that muscle pathology in MTM is reversible and likely amenable to therapeutic

intervention. In like manner to PIK3C2B, DNM2 downregulation holds immense promise as a

novel therapeutic strategy for MTM and potentially, all CNMs.

In 2011 Pierson et al. generated a murine model of the human c.205C>T point mutation (p.R69C)

in exon 4 of Mtm1 that has been consistently associated with milder forms of MTM40. Given the

short lifespan of Mtm1 KO mice, the existence of a milder model with a greater median survival

would be of benefit to the study of preclinical therapies, and the discovery of diagnostic windows.

Knock-in (KI) of the p.R69C missense mutation induced exon 4 skipping and the subsequent

generation of an out-of-frame transcript and premature stop codon in Mtm1. Notwithstanding the

complete loss of MTM1 in this model, Pierson and colleagues attributed the milder phenotypes

and increased lifespan (median survival= 66 weeks) of this model to the presence of several full-

length alternatively spliced forms of MTM1 in murine quadriceps40. This KI model has been used

in conjunction with the Mtm1 KO model described above to demonstrate that loss of Mtm1 is

associated with structural and functional abnormalities of the NMJ39,40.

Lastly, in 2013 Fetalvero et al generated the third Mtm1 KO model using a gene trapped Mtm1

allele41. Using this model, Fetalvero and colleagues speculated that loss of Mtm1 is associated with

impaired skeletal muscle autophagy, as evidenced by the presence of cellular abnormalities such

as ubiquitin aggregates, abnormal mitochondria and mTORC hyperactivation41. Interestingly, Al-

Qusairi et al found these aforementioned mechanisms to be impaired in the 129pas strain (Mtm1

KO)46. Interestingly, these findings are inconsistent with elevated PI(3)P levels, which should

over activate and/or promote excessive autophagy. Both Fetalvero et al and Al-Qusairi et al

speculate that loss of Mtm1 leads to an imbalance between mTOR and autophagy and therefore,

prevents the proper execution of autophagy and formation of autophagolysosomes41,46.

Anecdotally, our lab has been unable to identify any aberrant autophagic mechanisms in the Mtm1

KO mouse model. Not surprisingly, the in vivo study of autophagy is often complicated by the fact

the pathway is inherently complex and can be activated and/or inactivated as a consequence of a

myriad of intracellular and/or extracellular conditions. For this reason, further investigation and

13

increased scientific rigor is required to determine whether autophagic mechanisms are contributing

to, or responsible for MTM pathogenesis47,48.

5 Therapeutic Landscape of XLMTM At present, no clinically effective genetic and/or pharmacological therapies exist for MTM

patients. Nevertheless, the current approach to care and management relies heavily on a series of

multidisciplinary interventions aimed at improving survival and overall quality of life20. Most

novel therapeutic strategies undertaken to date have focused exclusively on the muscular re-

introduction of MTM1 by way of viral delivery or direct enzyme replacement therapy in MTM

murine models49,50. Pioneering of gene and enzyme replacement therapies is at present, an active

avenue of therapeutic development in MTM20. Recently, Childers et al. successfully delivered a

working copy of Mtm1 into both canine and murine models of MTM single injection of a muscle

trophic AAV8 vector49. In like manner, Lawlor et al. achieved short-term replacement of MTM in

murine models by intramuscular injection of a recombinant 3E10Fv-MTM1 protein replacement

agent. After two weeks of treatment this approach results in significant structural and functional

improvements in myopathic muscle50. Indeed, both groups have demonstrated the effectiveness of

targeted gene and enzyme replacement therapy in long-term improvements of MTM pathology;

specifically, in muscle strength and survival49,50. Nevertheless, the high cost of development and

administration that is associated with these novel yet promising approaches, coupled with the

present lack of clinical validation are reasons for which a strong rationale for identifying novel

therapeutic drugs persists. In light of this, our lab has recently undertaken various innovative

approaches to identify drug-targetable pathways in MTM.

Given that PI3P accumulation is a hallmark pathology of MTM skeletal muscle, we hypothesized

that genetic ablation of Pik3c2b, the PI3P generating muscle-specific class II PI3 kinase (PI3K),

would reduce the expression of PI3P and consequently, improve the pathology of the disease42.

We accomplished this by ablating Pik3c2b in Mtm1 KO mice, using both muscle-specific (Ckmm-

Cre), and tamoxifen-inducible Cre transgenic lines. The success of this study lead to our discovery

of Pik3c2b as a novel genetic modifier of Mtm1, given that Pik3c2b-/-Mtm1-/y double knock-out

mice displayed a complete phenotypic rescue and were remarkably indistinguishable from their

wildtype littermates42. The landmark success of this study set the stage for PI3K kinase inhibition

as a potential therapeutic strategy for MTM and other diseases of PIP metabolism. Despite being

the first study to provide promising preclinical evidence for PIK3C2B inhibition as a novel therapy

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for MTM, no class II PI3K inhibitors are currently undergoing clinical validation. Therefore, our

lab has pioneered several screens in silico and in vitro, as well as in zebrafish to further evaluate

the efficacy of available FDA-approved drugs and inhibitors specific to PIK3C2B to validate this

treatment strategy for clinical translation.

In a similar vein, following the identification of Dnm2 as another genetic modifier of Mtm125,26,

two groups have successfully reduced DNM2 using clinically relevant strategies44. Similarly,

Trochet et al. successfully reduced DNM2 protein and mRNA levels in murine and patient

fibroblast models of ADCNM using allele-specific silencing RNA (siRNA) against the ADCNM-

associated p.R465W mutation. This reduction was sufficient to achieve functional restoration in

both models of ADCNM51. These studies suggest that targeting DNM2 is an alternative and

potentially complementary therapeutic approach for MTM, and when taken in conjunction with

the therapeutic strategies mentioned above, support the existence of multiple disease mechanisms

in MTM that are amenable to therapeutic intervention.

Another approach involves the re-purposing of drugs that are currently in use for the treatment of

other conditions. Our lab has also undertaken this approach to identify novel therapies for MTM

that have established clinical translatability. Pyridostigmine is an FDA-approved

acetylcholinesterase inhibitor that is often used to treat congenital myasthenic syndrome52. By

virtue of similar clinical features between CNM patients and those affected with congenital

myasthenia, one case study aimed to identify whether fatigability and abnormalities in

neuromuscular transmission in patients with CNM are responsive to acetylcholinesterase inhibitor

therapy52. Of the four children examined in this study, only one was genetically diagnosed with

MTM and harboured a missense mutation (c.695A>G (p.His232Arg)) in exon 9 of MTM1. Not

surprisingly, this patient was affected with limited motor ability in childhood and became

wheelchair dependent by early adolescence. With pyridostigmine, the patient regained the ability

to stand and swim for uninterrupted and longer distances52. Further validation of pyridostigmine

in preclinical models was subsequently provided by our lab, using NMJ function and muscular

endurance as therapeutic outcome measures. Remarkably, both Mtm1 KO and Mtm1 KI mice

displayed significant improvements in grip strength and reduction in fatigability, as evidenced by

improved performance on the treadmill test following treatment with pyridostigmine22. These

findings correspond nicely with similar motor improvements in a morpholino knock-down

zebrafish model of MTM, which was also responsive to a-bungarotoxin, another

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acetylcholinesterase inhibitor22. Although these preclinical and clinical studies were the first to

identify a clinically established drug that may benefit MTM patients, pyridostigmine has since

showed only modest therapeutic benefit.

Of most notable importance towards the premise of my graduate work is our lab’s most recent

discovery of tamoxifen (TAM) as a potent mitigator of the hallmark pathologies of MTM. Briefly,

our findings unearth the first small molecule modifier of MTM that already possesses an excellent

clinical profile. Because TAM has already been approved by the FDA for use in the treatment of

estrogen receptor positive (ER+) breast cancer53, it is inexpensive and can be translated more

rapidly into the clinical arena. When compared to other novel therapeutic strategies, namely ASO-

mediated DNM2 knockdown and PIK3C2B inhibition, TAM is much less expensive to administer

and does not require extensive preclinical validation and refinement for safe use in humans. Re-

purposed drugs often do not face the significant hurdles encountered by other novel therapeutic

strategies before they can reach the patient population. Moreover, because TAM has a good safety

profile and is well-tolerated as a common adjuvant chemotherapeutic54, should clinical trials

confirm its effectiveness in MTM patients, it is possible that it could be used in conjunction with

various gene or enzyme replacement therapies to potentiate their effects. Furthermore, it is possible

that a novel drug therapy such as this would be potent enough to improve the quality of life and

survival of patients such that other, more expensive therapeutics are not required. Taken together,

recent advancements in the identification of novel therapies and their molecular targets in MTM

highlight the importance of identifying novel disease modifiers as a means to not only develop

more targeted therapeutic approaches, but to also broaden our understanding of the

pathomechanisms that drive this devastating disease. Consequently, the identification of pathways

underlying TAM’s therapeutic effect in MTM forms the basis of my graduate work and provides

the preclinical evidence necessary to accelerate its introduction into the clinical arena.

6 Pathophysiological Characterization of Mtm1 KO Mice and Mtm1 KO Mice Treated with Tamoxifen

As previously mentioned, the MTM mouse model used in this study was generated by the removal

of exon 4 using homologous recombination resulting in the complete loss of Mtm1 (Mtm1 KO)39.

At approximately 21 days (3 weeks) of age, Mtm1 KO mice begin to display progressively severe

muscle weakness and weight loss. Most importantly, Mtm1 KO mice possess a remarkably reduced

lifespan of approximately 36 days. By virtue of its faithful recapitulation of both the severity of

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the phenotypic and histopathological changes observed in the human disease, this particular model

has become one of the most extensively characterized murine models of MTM in the literature19.

Our discovery of TAM as a potential therapeutic for MTM occurred serendipitously during our

study exploring Pik3c2b ablation as a novel therapeutic strategy in MTM42. As an experimental

control for Cre-lox mediated Pik3c2b-/-Mtm1-/- double knockout mice, untreated Mtm1 KO mice

(non-Cre, non-floxed) were fed 40mg/kg of TAM daily, for one week. Interestingly, TAM-treated

Mtm1 KO mice displayed a moderate shift in their baseline survival from 35 days to 42 days42. To

validate this observation, we begun continuously treating Mtm1 KO mice at 21 days of age with

two different doses of TAM (3mg/kg/day (low) and 40mg/kg/day (high)) formulated in their food

chow, or standard chow (placebo). Remarkably, we observed that long-term TAM treatment

resulted in significant improvements in median survival to 57 days at high dose and 48 days at low

dose. At the pathophysiological level, both doses of TAM increased the grip strength of Mtm1 KO

mice to levels equivalent to those of wild-type (WT) littermate controls. Most notably, this

observation was coupled with significant improvements in sacrotubular and membrane structures.

Interestingly however, major histopathological improvements were seen exclusively with high

dose TAM, namely a significant reduction in the abundance of centralized nuclei and increase of

myofiber size. Remarkable improvements in triad number, structure and function were also

exclusive to the effect of high dose TAM. Overall, our data elegantly demonstrates that TAM

provides an exceptional therapeutic benefit to Mtm1 KO mice and acts in a dose-dependent manner

to rescue the structural and functional integrity of skeletal muscle. Understanding the nature of this

dose-dependent modulation is important for identifying whether different pathomechanisms

underlie the structural and functional abnormalities we observe in myopathic muscle. Given that

3mg/kg/day is reminiscent of doses used in pediatric settings, further experimentation and dosing

strategies will promote the clinical translatability of TAM and introduce the possibility of

combinatorial therapeutics as a feasible approach to modulate different aspects of the disease. This

dose-dependent effect is further reinforced through clinical trials reporting that “high” doses

(80mg/day to 720mg/day) of TAM are well-tolerated in humans and are effective for the treatment

of non-breast cancers such as glioma, melanoma and lung cancer54. Most notably, these doses are

considerably higher than the dose required to inhibit estrogen receptors (20mg/day)54, and

consequently suggests that TAM exhibits a therapeutic suitability beyond that of the treatment of

ER+ breast cancer. This supports our rationale for identifying the molecular sequence of events

underlying TAMs therapeutic benefit in myopathic muscle as a means to uncover the etiology of

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hallmark muscular abnormalities in MTM and in doing so, identify novel disease contributors that

may in part explain the pathogenesis of MTM.

7 Estrogen Signalling as a Modulator of Skeletal Muscle Structure and Function

Estrogens function to regulate cellular growth and differentiation within all tissues, and more

specifically within those of the reproductive system55. The systemic effects of estrogens occur

through the activation of estrogen receptor alpha (ERa) and estrogen receptor beta (ERb). Once

bound, 17b-estradiol promotes the recruitment of receptor-specific transcriptional factors and

coactivators that regulate the expression of several downstream ER-related genes, many of which

are involved in cellular growth, development and metabolism56. In addition to the well-

documented effect of estrogen-dependent signalling in the development of bone and reproductive

tissues, the biological significance of this signalling pathway within other tissues and organ

systems has become increasingly understood55,57,58. Mice lacking ubiquitous expression of ERa

(Esr1 KO) display pathologies reminiscent to that of a metabolic syndrome with functional defects

in a variety of tissues, namely: impaired oxidative metabolism, insulin resistance, inflammation,

impaired glucose tolerance increased body fat and body mass, to name a few59–62. Consequently,

modulating the actions of ERa and ERb constitutes a popular therapeutic approach for various

estrogen-related diseases and most notably, cancer.

TAM is the most common nonsteroidal selective estrogen receptor modulator (SERM) that is used

in the treatment of ER+ breast cancer and other estrogen-related diseases63. As a structural

analogue of 17b-estradiol, TAM competes with ER ligands (mainly 17b-estradiol) for binding to

ERa or ERb. Depending on the tissue and cellular context within which this interaction occurs,

TAM regulates the expression of different genes as either an antagonist or agonist of either

receptor63,64. TAM functions to recruit tissue and cell-specific transcriptional factors and

coactivators to induce downstream transcriptional signatures that are receptor-specific and

moreover, correspond to the particular tissue type and cellular environment within which TAM is

acting. In this way, TAM modulates vital cellular processes such as cell growth, development,

differentiation and overall homeostasis63,64. This differential activity of TAM permits its dual use

as both an anti-estrogenic adjuvant chemotherapeutic for ER+ breast cancer, and estrogenic

therapeutic for osteoporosis54. This is in contrast to fulvestrant, a structurally dissimilar estrogen

receptor antagonist that once bound to ERa, accelerates its complete degradation63. In this way,

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fulvestrant acts to completely inhibit ERa signalling and downstream transcriptional signatures.

Not surprisingly, fulvestrant is commonly used as an alternative therapeutic for TAM-resistant and

recurrent forms of ER+ breast cancer65.

Intriguingly, ER signaling has been recently implicated in the modulation of skeletal muscle

structure and function57. Most notably, females with reduced circulating levels of estrogen exhibit

a significant reduction in muscle strength and increased incidence of muscular atrophy66. Similarly,

mice lacking the muscle-specific expression of ERa share similar phenotypes59,62,67. Not

surprisingly, estrogen signalling is advantageous for maintaining the biological integrity of skeletal

muscle, through protection against oxidative stress and contraction-induced injury62,68–71. In line

with this observation, hormone replacement therapy has been shown to reintroduce estrogenic

benefits in post-menopausal women; resulting in improved force generation and structural

integrity62,66. Of most relevance to the premise of my graduate work, is recent evidence implicating

17b-estradiol and TAM as transcriptional modulators of ER-dependent genes in skeletal muscle64.

In keeping with the fact that mechanisms of ER signaling are inherently complex, the exact manner

by which this modulation occurs in the context of skeletal muscle and particularly male skeletal

muscle, remains to be fully understood. This however, presents a unique opportunity for

therapeutic discovery. In support of this, a recent study by Dorchies et al, was the first to

demonstrate the ability of TAM to improve muscular pathology in a murine model of Duchenne

muscular dystrophy (DMD)72. Moreover, our lab’s serendipitous but landmark discovery of

TAM’s therapeutic benefit in MTM further reinforces the possibility that the dysregulation of

estrogen-related genetic and/or molecular signatures may underlie distinct muscular pathologies

in MTM and other muscle disorders. Identifying the mechanism of TAM’s therapeutic effect in

MTM will not only help clarify our understanding of ER signaling in skeletal muscle, but also

whether the existence of any estrogen-related pathologies connect the hallmark molecular

pathologies of MTM (i.e. the accumulation of PtdIns(3)P and DNM2) to structural abnormalities

in muscle. It is also possible that abnormalities in the accumulation of PtdIns(3)P and DNM2 occur

secondarily to, or as a consequence of the dysregulation of a more global pathway that modulates

the overall biology of skeletal muscle.

8 Proteostasis as a Mechanism of Skeletal Muscle Maintenance In conjunction with ER signaling, the ubiquitin-proteasome system (UPS) is another molecular

mechanism that functions to maintain the overall structure and function of skeletal muscle73. As a

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primary mechanism of protein turnover in mammalian cells, the UPS functions to exclusively

remove misfolded and/or damaged intracellular proteins, whereas autophagic mechanisms

promote the turnover of larger cytosolic aggregates74. Not surprisingly, the UPS regulates a variety

of cellular processes such as the cell cycle and stress response. Given that muscle cells harbour a

heightened sensitivity to proteotoxic stress, the UPS is particularly important for the maintenance

of structural integrity within muscle fibers73,75. The dysfunction of this system is causative of a

class of neuromuscular diseases known as proteinopathies, in which vital cytoskeletal proteins are

misfolded and aggregated within affected muscle cells73.

Interestingly, ERa and DNM2 are both regulated by the UPS; where in the specific case of ERa,

the proteasome mediates estradiol-induced negative feedback mechanisms to regulate ERa levels

in keeping with the extracellular supply of estrogen74,76,77. Most notably, both 17b-estradiol and

TAM have been recently implicated as modulators of the proteasome, the activity of which

significantly impacts the expression and biological function of many proteins73,74,77. In support of

this, Kuo et al. demonstrated that TAM decreased the DNA repair activity of O(6)-methylguanine

DNA methyltransferase (MGMT) in HT-29 carcinoma cells by accelerating its proteasomal

degradation. Interestingly, this effect occurred independently of transcriptional changes and the

intracellular expression of ERa78. Even more striking, are findings from Gavriilidis et al. that

implicate MTM1 as a modulator of protein homeostasis for the first time. Briefly, MTM1 and

ubiquilin-2 (UBQLN2) were discovered to function as a complex that triggers the cytosolic

degradation of intermediate filament proteins desmin and vimentin75. This suggests that the

degradation of cytoskeletal proteins in muscle cells prior to misfolding or aggregation is subject to

tight regulation. Moving forward, this novel and possibly PtdIns(3)P-independent role of MTM1

as a mitigator of proteotoxic aggregate formation in skeletal muscle is of great relevance to

uncovering the mechanism of action of TAM in MTM. It is possible that TAM functions in MTM

by way of a mechanism that is independent of its traditional transcriptional role as a breast cancer

therapeutic. Moreover, should TAM be found to modulate a global pathway like the UPS, it would

correspond nicely with correction of molecular pathologies in Mtm1 KO mice that are both related

and unrelated to the function of ERs.

It is also possible that TAM modulates autophagy in MTM. Interestingly, both Al-Qusairi et al.

and Fetalvero et al. show that loss of Mtm1 is associated with impaired autophagy and proteasomal

20

degradation in two different MTM mouse models41,46. However, determining whether autophagic

defects underlie the primary cause of MTM, and moreover whether the therapeutic effects of TAM

are a consequence of autophagic modulation warrants further investigation. Despite recent

advances in our understanding of the molecular aspects of autophagy, the ability to effectively

study it in vivo however, is subjective and further complicated by nature of its broad roles in health

and disease. Furthermore, drugs that either inhibit or activate autophagy (i.e. mTOR inhibitors)

also have effects in unrelated pathways (i.e. protein, and lipid synthesis)47. Consequently, it not

only becomes difficult to evaluate the effectiveness of these drugs in vivo, but also to ascertain

whether autophagy is necessary for protection against disease, or whether its defects are

responsible for the improvement in disease pathogenicity. It is for this reason that many studies

present conflicting evidence. Evidently, autophagy is highly dependent on intracellular and

extracellular conditions, therefore effectively turning it on and off whilst controlling for

cofounding pathways requires further experimental refinement and investigation47,48. It is for this

reason, that investigation into the autophagic contributions to MTM and more specifically, the role

of TAM as a modulator of this pathway lay beyond the time constraints of my graduate work.

9 Summary

Based on the information presented above, the specific aims of my Master’s Thesis were: 1) to

identify whether TAM was acting dependent or independent of the ER in Mtm1 KO mice, 2) to

validate the clinical feasibility of TAM as a novel therapeutic for MTM by identifying changes in

the expression of genetic and/or molecular signatures that associate with improved phenotypes

observed Mtm1 KO mice following TAM treatment. Towards the completion of my first aim, my

collaborator Nesrin Sabha, focused exclusively on the pathophysiological characterization of

Mtm1 KO mice, as well as the identification of the unique histopathological improvements in

Mtm1 KO mice following TAM treatment. Using a variety of phenotypic and histological

assessments she established hallmark structural and functional abnormalities within skeletal

muscle of Mtm1 KO mice and in doing so, defined prognostic features of the murine disease. She

then characterized the remarkable ability of TAM to rescue the aforementioned pathologic

abnormalities in MTM muscle. Most notably, she observed significant improvement in the activity,

overall strength and median survival of Mtm1 KO mice following treatment. Furthermore, our

collaborator Robert Dirksen at the University of Rochester, performed all the functional studies on

triad pathology (twitch and tetanic stimulation), by measuring electrically evoked Ca2+ release in

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single flexor digitorum brevis muscles following individual isolation from Mtm1 KO mice and

TAM-treated Mtm1 KOs.

As a well-documented modulator of ER activity, TAM is known to modulate estrogen receptor

signalling by directly binding to either ERa or ERb within different tissues and cellular contexts63.

In light of this, I first explored whether TAM’s primary mechanism of action in MTM is by way

of ER modulation, and/or modulation of well-established effectors of known ER pathways. Given

that ER signalling is inherently complex, and not well characterized within the context of male

skeletal muscle, this study is the first of its kind to establish whether TAM exploits this pathway

to improve the pathophysiology of a MTM. Moreover, should TAM function by way of a novel

mechanism, be it specific or non-specific to skeletal muscle, it would have several important

implications, namely the exploitation of its therapeutic potential beyond ER+ breast cancer.

My second aim is based on the reasonable expectation that the therapeutic effects of TAM in MTM

are a consequence of discrete genetic and/or molecular changes. Furthermore, these signatures may

be novel, or downstream of known ER pathways. Indeed, TAM has been reported to function

independently of the ER as a modulator of several cellular pathways. In support of this, Daurio et

al demonstrated that TAM acts to regulate tumor metabolism through AMPK activation,

independently of ER. Moreover, TAM was able to induce cell death in both ER+ and ER- breast

cancer cells, and triple-negative breast cancer cells54. In order to characterize the nature and

sequence of molecular events that occur following TAM treatment in Mtm1 KO mice, I have

performed a series of drug screens in various mammalian cell lines. Through this approach, I have

identified a pathway through which TAM is likely to induce the hallmark molecular improvements

observed in Mtm1 KO mice. A parallel in vitro study was done by another member of our lab to

further investigate the estrogen-receptor dependence of TAM, the results of which will be

discussed in Chapter 4. Consequently, the characterization of the molecular mechanism of this

drug and validating its use for clinical translation in MTM patients forms the basis of my graduate

work. To my knowledge, these experiments comprise the first pre-clinical validation of an FDA

approved drug with potential for clinical translation as a small-molecule modifier of MTM.

Taken together, it is clear from the information presented above that it is important to understand

the molecular sequence of events that underlie the development of this devastating disease, and

particularly whether disease-driving pathomechanisms are the same those that are preferentially

22

modulated by TAM. In this way, I aim to validate the therapeutic potential of TAM for the

treatment of MTM. Perhaps one of the most impactful consequences of this work is the support it

lends towards drug repurposing. This approach has immense potential to shed insight into novel

implications of basic and well-established cellular mechanisms in the pathogenesis of diseases that

have yet to be fully understood.

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Chapter 2 In-Vivo Analyses of the Expression and Localization of Estrogen Receptors

in Mtm1 KO mice and Tamoxifen-treated Mtm1 KO mice

Towards the completion of this chapter, my colleague, Nesrin Sabha, has successfully established

the characteristic pathology of the Mtm1 KO mouse model and identified the most notable

histopathological changes in Mtm1 KO mice treated with TAM, 17b-estradiol and fulvestrant.

Unless otherwise stated, the data presented hereafter will focus exclusively on my investigation

into the unique molecular signatures underlying the Mtm1 KO phenotype, as well as on those

associated with the therapeutic effects of TAM. For the pathophysiological characterization of

Mtm1 KO mice treated with 17b-estradiol and fulvestrant, Nesrin Sabha and I worked

collaboratively to complete the indicated experimentation.

10 Expression of Estrogen Receptors

The classic mechanism of ER signalling occurs when 17b- estradiol binds to either ERa or ERb

in the nucleus, after which receptor dimerization initiates and promotes binding of the receptor to

estrogen-response elements (EREs) in the regulatory regions of ER-response genes79. Although

this is the most well-known mechanism of estradiol-induced gene regulation, ERs can also regulate

gene expression by two other indirect mechanisms, one of which involves recruitment of

transcription factors in the nucleus and the other, activation of a series of non-genomic signalling

pathways as a plasma membrane-bound receptor79,80. These latter two mechanisms, along with

specialized domain structures of each ER subtype, are what enable ERs to regulate a variety of

ER-response genes in an ERE-independent, genomic or non-genomic manner. For example, ERs

often recruit AP-1 transcription factor complexes in the nucleus through their AF-2 domain to

regulate the expression of cyclin D1 and IGF-I, among others79. Moreover, ERs (amongst other

nuclear hormone receptors) may function as membrane-bound receptors to activate extra-nuclear,

non-genomic activity of several protein-kinase signalling cascades (i.e. MAPK, PI3K-AKT); these

interactions are thought to be mediated by ligand-binding domain (LBD) of ERs81,82, however the

exact nature of this non-genomic modulation is unknown and under extensive investigation.

It is evident that ER-mediated regulation of gene expression is complex as it includes three distinct

signalling modalities that are either genomic or non-genomic. It is also important to note that the

24

final transcriptional signature that is induced following estradiol-mediated activation of ERa or

ERb, may depend on conditions. The relative tissue expression of ERa or ERb is one such

condition that can determine how a tissue or cell responds to estrogens56. Contrary to the

proliferative role of ERa, ERb is believed to exert anti-proliferative effects in bone and

reproductive tissues79,83. Other conditions include the nature of the ligand (i.e. 17b-estradiol,

SERMs, SERDs), the combination of transcription factors that are recruited, and the abundance

and type of downstream coregulatory proteins and components of intracellular signalling

pathways63. These factors moreover, are highly dependent on the cell type and/or tissue within

which ER subtypes are expressed. Once bound to a particular ligand, ERa and ERb possess the

molecular flexibility to evoke unique transcriptional signatures as homodimers or heterodimers by

way of genomic or non-genomic signalling modalities in a variety of cellular contexts56. This is

further complicated by the fact that both ERa and ERb are subject to alternative splicing to produce

protein-coding receptor isoforms that lack certain domains and consequently regulate genes by

way of different mechanisms81,84. Two classic examples are the well-characterized splice variants

of ERα, ERα46 and ERα36, in which both or one of the transcriptional activator functions (AF

domains) are lacking80–82. For this reason, mechanisms by which either isoform functions to

regulate transcription differ from that of the full-length receptor.

ER-responsive genes and genes downstream of ER-regulated signalling pathways can greatly

influence the overall physiology of tissues beyond those involved in the development of

reproductive cancers. Many studies have pinpointed the biological importance of estrogens and

their downstream targets in non-reproductive tissues such as skeletal muscle62,64,68. This is

particularly important given that the exact mechanism(s) by which ER-induced gene regulation

occurs within the context of skeletal muscle, specifically in males, is poorly understood.

Understanding mechanisms governing ER signalling in skeletal muscle will help to advance our

understanding on the contribution of this pathway towards the development of neuromuscular

diseases. This is an important strategy that may advance the development of therapies against

specific receptors and/or receptor targets in complex diseases such as MTM.

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In light of the well-established role of TAM as a transcriptional modulator of the ER, I first sought

to determine the expression profile of ERα and/or ERβ in skeletal muscle of Mtm1 KO mice. By

Western blot, I was able to detect the expression of two distinct isoforms of ERα, the well-

characterized “wild-type” 66kDa isoform (ERα66) and a shorter 55kDa (ERα55) isoform, in Mtm1

KO mice (Figure 4A,D). Furthermore, I detected a significant increase in the expression of ERα55

in Mtm1 KO mice compared to wild-type controls (p= 0.002) (Figure 4C). and (p= 0.0049)

(Figure 4F) , whilst expression levels for ERα66 remain unchanged (Figure 4B,E). Given that the

expression of ERs in human and murine skeletal muscle has been shown to be lower in comparison

to other tissue types, it is particularly surprising to see significant enrichment of a specific ERα

isoform in the skeletal muscle of Mtm1 KO mice and most strikingly, in male skeletal muscle.

Remarkably, following exposure to high and low dose TAM treatment, I was able to demonstrate

that both doses induced a significant reduction in ERα55 expression compared to untreated Mtm1

KO mice (p= 0.0112) and (p= 0.0097), albeit remaining moderately high in comparison to wild-

types. (Figure 4C, F). This is in contrast to the expression levels of ERβ, which were minimal and

remained unchanged across all experimental conditions (Figure 5). This suggests that there is a

specific biological consequence of ERa upregulation in MTM muscle, and moreover that the

phenotypic improvements we observe following TAM treatment may be due to the specific

modulation of ERa, rather than that of ERs in general.

Figure 4. High and low-dose TAM treatment reduces ERα expression in vivo. (a,d) Representative Western blots for ERα with b-actin loading control, for WT, WT+TAM, Mtm1 KO, and TAM-treated Mtm1 KO mice. Molecular weight markers indicated in kDa. Samples for each drug trial derive from the same experiment and all blots were run in parallel for each trial. Protein levels of both the 66kDa and 55kDa isoforms of ERα as determined by densitometry for (b,c) high-dose TAM and (e,f) low-dose TAM, standardized to b-actin and represented as fold difference from the average of WT ± SEM (n=5 mice per group for high dose TAM, n=3 mice per group for low dose TAM. All statistical analyses were conducted

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by two-way ANOVA, followed by Tukey’s multiple comparisons post-test. *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. Given that ERα can be expressed as many functional splice forms81, I sought to determine whether

this 55kDa protein isoform of ERα has been previously characterized and if so, what functional

information may shed light into the biological significance of its upregulation in Mtm1 KO mice.

Following an extensive search of the literature search for reports and/or characterization of this

isoform in previous studies, I was unable to confirm the identity of this band despite extensive

literature on the different splice forms of ERa and their predicted molecular weights81,85,86. The

antibody I used to detect ERa by Western blot is rabbit polyclonal and was manufactured by Santa

Cruz (SC-542, Clone MC-20); it is therefore highly likely that this 55kDa band is in fact an isoform

of ERa and not simply an unspecific endogenous murine protein. This particular clone (MC-20)

has been extensively used in over 300 publications and is said to be raised against an unspecified

peptide region in the c-terminus of murine ERa. This antibody, in addition to all other Santa Cruz

antibodies that have been used in this project, have since been discontinued by the company. In

fact, the company has recently discontinued the majority of its antibodies and replaced them with

newly synthesized alternatives. Unfortunately, the only ERa alternative (SC-8002, Clone F-10)

that is currently available from Santa-Cruz can only detect protein of human origin. When I

assayed this antibody against ERa in murine muscle, I was unable to visualize any bands. Given

that murine ERa spans 595 amino acids, it is possible that this unidentified 55kDa isoform is

comprised of a novel peptide sequence that has yet to be characterized.

As a future direction, at the protein level, a logical next step would be to conduct a series of

Western blots using various ERa antibodies that have been raised against different peptide regions.

For example, Western blots of quadriceps of wild-type and MTM mice should be run in parallel

and visualized using the MC-20 clone and other antibodies that have been raised against either the

N-terminal region, or ERα peptides comprised of amino acids closer to the N-terminal region of

the protein. Moreover, an antibody can be used that is only specific to detect the 66kDa isoform to

the exclusion of others (i.e. ERα36 and ERα46). This would be a feasible technique with which to

determine the isoform size or region that is missing in ERα55 using a process of elimination.

Although this strategy may not directly indicate the sequence composition of ERα55, it would be

a good way to determine whether ERα55 is in fact an ERα isoform and a general idea of which

domains/amino acid regions are lacking in this particular isoform. These findings could then

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provide a guideline within which a more comprehensive study can be designed to determine the

peptide and/or cDNA sequence of the 55kDa band. Moreover, by looking into the expression of

ERα in other tissues and muscle types can help determine whether the 55kDa band is muscle

specific and moreover, whether the expression of this isoform is inherently increased in all ERα-

expressing tissue types of MTM mice.

Figure 5. The effect of high-dose TAM on the expression of ERß. Representative Western blot for ERß (a), with b-actin as a loading control; position of the molecular weight markers indicated (in kDa). Both protein isoforms can be seen at approximately 66kDa and 55kDa. Protein levels of both the 66kDa (b) and 55kDa (c) isoform were determined by densitometry and standardized to b-actin. Graphs represent five biological replicates and the fold difference from the average of the WT ± SEM. (n=5 mice per group). Unlike ERa, there is no significant differences in the expression of either isoform of ERß between treatment groups. All statistical comparisons were conducted by two-way ANOVA, followed by Tukey’s multiple comparisons post-test.

Only one 55kDa isoform of ERα is reported in Ensembl (ENSMUST00000105588.7) and UniProt

(D3Z6V3) with a 3’ sequence unique from that of the well-characterized 66kDa isoform

(ENSMUST00000105590.7; P19875). All other regions of this annotated 55kDa isoform are

identical in sequence to that of the 66kDa isoform. According to UniProt, the protein sequence of

the 55kDa isoform spans from amino acid 10 to 490. To determine whether the isoform that is

enriched in Mtm1 KO muscle is identical to the annotated one, I designed 3 sets of primers for

each isoform that were complementary to the 3’ sequence of the 66kDa isoform and the unique 3’

sequence of the annotated 55kDa isoform. I then investigated whether I could detect the ERα

mRNA in the quadriceps of Mtm1 KO mice by reverse transcriptase-PCR (RT-PCR). Despite

being able to successfully detect the 66kDa isoform of ERα, I was unable to successfully isolate

this 55kDa isoform based on the reported sequence (Figure 6). Although it is possible that my

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primer sets were not specific enough or required further optimization, it is also likely that this

isoform has not been well-characterized. Consequently, I carefully excised the bands

corresponding to both the 66kDa and 55kDa ERα isoforms from a well separated SDS-PAGE gel

and sent both bands from WT and Mtm1 KO mice to the SPARC BioCentre at the Hospital for

Sick Children for identification by mass spectrometry. Unfortunately, results gleaned from this

experimental strategy were non-specific in that neither ERα66 or ERα55 were identified as

abundant proteins in quadriceps samples from either WT or Mtm1 KO mice. This inconsistent

result may have occurred for a variety of reasons. For example, more abundant proteins (i.e.

globulins) have the same molecular weight (50-70 kDa) as both isoforms and may therefore

masked the expression of other less abundant peptides. The success of this experiment highly

depends on my ability to effectively conduct the crude isolation of both bands of interest from an

SDS-PAGE gel by eye, to the exclusion of any surrounding proteins. This therefore represents a

likely disadvantage of this strategy, and it may in part explain why I was unable to accurately

detect or determine the peptide sequence of either isoform by mass spectrometry.

Figure 6. RT-PCR mediated isolation of ERα66 and ERα55 mRNAs. Two of the three sets of primers against the 3’ sequence of ERα 66 were successful in isolating a 180bp amplicon. Neither primer set designed specifically against the unique 3’ cDNA sequence of the annotated 55kDa ERα isoform were successful in isolating the predicted 171 base-pair amplicon. This suggests that the 55kDa isoform of ERα that I have observed by Western blot is not the same as the 55kDa protein coding isoform of ERα annotated in Ensembl and Uniprot.

In order to determine whether ERα upregulation in Mtm1 KO mice was a consequence of isoform-

specific transcriptional dysregulation, and whether TAM was acting as a transcriptional modulator

of ERα, I investigated the expression levels of ERα mRNA in all four experimental conditions by

real-time quantitative PCR (qPCR). I designed primers for ERα that spanned genetic regions that

were unaffected by alternative splicing using Primer 3 Input (Version 0.4.0). For comparison, I

also conducted the same analysis for ERb. Contrary to protein expression, ERa mRNA levels were

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significantly decreased in Mtm1 KO mice (p= 0.0048) compared to controls (Figure 9A). This

conflicting result likely suggests that ERa transcription is decreased to offset the increased

expression of the receptor in Mtm1 KO mice.