Defining Variant Function and Therapeutic Targets in Pediatric Cancer Restricted; Files Only

Abstract

Pediatric cancers arise primarily from developmental disruptions rather than accumulated mutations, creating unique challenges for interpreting rare variants and identifying actionable therapeutic targets. We address these challenges through complementary functional genomics approaches in two clinically important contexts: SMARCB1-deficient tumors and Wilms tumor.

We performed comprehensive deep mutational scanning of SMARCB1, encompassing 8,418 amino acid substitutions across the entire coding region to assess their functional impact. This revealed that missense mutations in the RPT2 domain of SMARCB1 disrupt antiproliferation function by destabilizing the SWI/SNF complex and impairing chromatin remodeling and transcriptional regulation to an extent comparable to nonsense mutations. Structural modeling demonstrated that these variants destabilize the hydrophobic core of the RPT2 domain through disruption of van der Waals interactions. Critically, these loss-of-function missense variants maintain detectable protein expression, challenging current diagnostic reliance on immunohistochemistry and highlighting limitations of existing mutation classification approaches.

To identify therapeutic vulnerabilities in Wilms tumor, we established twelve patient-derived cell lines that faithfully recapitulate tumor biology. We then performed targeted loss-of-function screens in a subset of these lines to identify overlapping dependencies. These screens revealed nuclear export machinery, specifically XPO1, as a selective dependency. Treatment with the FDA-approved XPO1 inhibitor KPT-330 suppresses expression of TRIP13, a mitotic checkpoint regulator required for Wilms tumor cell survival. We further identified synergy between KPT-330 and doxorubicin, a chemotherapy used in high-risk favorable histology Wilms tumor, which leads to durable remissions in vivo.

Together, these studies address two critical gaps in pediatric precision oncology: functional interpretation of variants in low-mutation-burden cancers and rational identification of therapeutic dependencies. By integrating high-throughput functional assays with genetic perturbation screens, this work advances our ability to translate genomic findings into biological understanding and therapeutic opportunities for children with cancer.

Table of Contents

Chapter 1. Introduction

1.1 Pediatric Cancer: Developmental Origins and Clinical Challenges...............................1

1.2 Advances in Cancer Genomics: From Sequencing to Functional Interpretation........ ....3

1.2.1 Cancer Mutation Databases and the Challenge of Data Integration.......................3

1.2.2 Computational Prediction of Variant Pathogenicity......................................... ....5

1.3 Functional Genomics Approaches for Variant Validation and Target Discovery..............7

1.3.1 Deep Mutational Scanning: Comprehensive Functional Maps of Protein Variants...8

1.3.2 Loss-of-function Screens for Therapeutic Target Discovery................................. ....10

1.4 SMARCB1-Deficient Tumors: Chromatin Remodeling Gone Awry................................12

1.4.1 The SWI/SNF Chromatin Remodeling Complex...................................................12

1.4.2 Clinical Spectrum and Molecular Characteristics.................................................13

1.5 Wilms Tumor: Disrupted Development and the Search for New Targets.......................14

1.6 Dissertation Overview and Aims...............................................................................17

 

Chapter 2. The Role of SMARCB1 in Cancer: An Overview

2.1   Abstract................................................................................................................21

2.2   Introduction..........................................................................................................21

2.3   SMARCB1-Deficient Cancers...................................................................................24

2.3.1   Types of SMARCB1-Deficient Cancers...............................................................25

2.3.2   Initiation of SMARCB1-Deficient Tumors..........................................................25

2.3.3   Unique Role of SMARCB1 in Synovial Sarcoma...................................................27

2.4   Structure of SMARCB1............................................................................................28

2.4.1   Winged Helix Domain......................................................................................29

2.4.2   Tandem Repeat (RPT) Domains........................................................................30

2.4.3   C-terminal Coiled-Coil Domain (CTD)...............................................................31

2.4.4   Conservation of SMARCB1................................................................................31

2.5   SMARCB1 and the SWI/SNF Complex.......................................................................32

2.5.1   Three Different BAF Sub-Complexes..................................................................33

2.5.2   Transcriptional Regulation by SMARCB1............................................................33

2.5.3   SMARCB1 Regulation of Super Enhancers...........................................................35

2.5.4   SMARCB1-Dependent BAF Complex Stability......................................................36

2.6   Molecular Functions of SMARCB1.............................................................................37

2.6.1   SMARCB1 Acting as a Tumor Suppressor via Regulation of p16INK4a....................37

2.6.2   SMARCB1 Inhibits MYC Target Activation...........................................................37

2.6.3   Exportin 1 (XPO1)-Mediated Localization of SMARCB1........................................38

2.7   Advances in Molecular Subgrouping of ATRT..............................................................39

2.8   Therapeutic Vulnerabilities of SMARCB1-Deficient Cancers........................................42

2.8.1   Opposing Overactive PRC2 Repression by Inhibiting EZH2...................................43

2.8.2   Targeting GBAF Dependency through BRD9 Degradation.....................................44

2.8.3   Tyrosine Kinase Inhibition (PDGFRα/β and FGFR2).............................................44

2.8.4   MYC Inhibition.................................................................................................45

2.8.5   Immune Checkpoint Inhibition..........................................................................45

2.8.6   High-throughput Screens to Identify Therapeutic Vulnerabilities.........................45

2.8.7   MDM2/4 Inhibition...........................................................................................46

2.8.8   Proteasomal Inhibition......................................................................................46

2.9   Conclusions.............................................................................................................48

 

Chapter 3. SMARCB1 missense mutants disrupt SWI/SNF complex stability and remodeling activity

3.1 Abstract.....................................................................................................................52

3.2 Introduction...............................................................................................................52

3.3 Results.......................................................................................................................55

3.3.1 Mutational landscape and functional diversity of SMARCB1 variants.......................55

3.3.2 Deep mutational scanning of SMARCB1 coding sequence....................................... 59

3.3.3 Functional scores of 8,418 SMARCB1 mutants........................................................61

3.3.4 Functional and structural insights in mutation-intolerant regions of SMARCB1.......64

3.3.5 Missense mutations in SMARCB1 disrupt SWI/SNF complex integrity......................66

3.3.6 Missense mutants disrupt attractive van der Waals forces in hydrophobic core of  RPT2     

..................................................................................................................................69

3.3.7 SMARCB1 missense mutations selectively disrupt distal enhancer regulation..........73

3.4 Discussion.................................................................................................................80

3.5 Supporting Data.........................................................................................................86

3.6 Methods.....................................................................................................................100

 

Chapter 4. Targeting TRIP13 in favorable histology Wilms tumor with nuclear export inhibitors synergizes with doxorubicin

4.1 Abstract......................................................................................................................126

4.2 Introduction................................................................................................................126

4.3 Results........................................................................................................................127

4.3.1 WT cell lines faithfully recapitulate genomic and transcriptomic features of WT............127

4.3.2   RNAi and CRISPR-Cas9 screens identify XPO1 as a potential therapeutic target in WT..131

4.3.3   XPO1 inhibition induces cell death through the TRIP13/p53 axis.................................135

4.3.4   KPT-330 and doxorubicin are synergistic in vitro and in vivo in WT..............................141

4.4 Discussion....................................................................................................................145

4.5 Supporting Data............................................................................................................149

4.6 Methods........................................................................................................................160

 

Chapter 5. Discussion and Future Directions

5.1 Introduction...................................................................................................................170

5.2 SMARCB1 Variant Interpretation: From Functional Data to Clinical Utility..........................170

5.2.1 Deep Mutational Scanning Enables Comprehensive Variant Interpretation in SMARCB1

.......................................................................................................................................170

5.2.2 SMARCB1’s Mutational robustness reflects biological architecture...............................175

5.2.3 The Problematic Use of Mutation Frequency as a Proxy for Pathogenicity.....................176

5.2.4 Context-Dependent Pathogenicity: The R377H Case Study..........................................179

5.2.5 Hypermorphic Variants: An Underexplored Dimension................................................182

5.3 Therapeutic Vulnerabilities in Wilms Tumor......................................................................183

5.3.1 Overcoming Model System Limitations Through Focused Screening.............................183

5.3.2 Nuclear Export as a Critical Dependency.....................................................................184

5.3.3 Convergent Dependencies Across Pediatric Kidney Cancers..........................................185

5.4 Methodological Considerations and Future Directions........................................................187

5.4.1 The Need for In Vivo Validation..................................................................................187

5.4.2 Computational Approaches to Complement Experimental Genomics............................187

5.5 Closing Remarks - Functional Genomics as a Bridge...........................................................190

References............................................................................................................................193

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Genetics and Molecular Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Bioinformatics Health Sciences, Oncology Biology, Genetics
Keyword	Genetic variants Genetic sequencing Wilms tumor Functional genomics SMARCB1 Therapeutic targets Pediatric cancer
Committee Chair / Thesis Advisor	Andrew Hong, Emory University
Committee Members	Carlos Moreno, Emory University David Katz, Emory University Roger Deal, Emory University Karen Conneely, Emory University

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