Characterization of MYC Interaction with Nuclear Receptor SET Domain Protein 3 (NSD3) Open Access

Gonzalez-Pecchi, Valentina (Spring 2019)

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As genomics advances reveal the cancer genome landscape, a daunting task is to understand how these genes contribute to dysregulated oncogenic pathways. Integration of cancer genes into networks offers opportunities to reveal protein-protein interactions (PPI) with functional and therapeutic significance. The generation of a cancer-focused PPI network termed OncoPPi, identify 397 cancer-associated PPIs. PPI hubs reveal new regulatory mechanisms for cancer genes like MYC.

 The MYC transcription factor plays a crucial role in cell growth control. Enhanced MYC protein stability has been found to promote tumorigenesis. Thus, understanding how MYC stability is controlled may have significant implications for revealing MYC-driven growth regulatory mechanisms in physiological and pathological processes. Our previous work identified the histone lysine methyltransferase Nuclear Receptor SET Domain protein 3 (NSD3) as a MYC modulator. NSD3S, a non-catalytic isoform of NSD3 with oncogenic activity. However, the mechanism by which NSD3S regulates MYC remains to be elucidated. To uncover the nature of the interaction and the underlying mechanism of MYC regulation by NSD3S, my research revealed that NSD3S binds, stabilizes, and activates the transcriptional activity of MYC. Further characterization of the binding interface between both proteins narrowed the interface to a 15 amino acid region in NSD3S that is required for MYC regulation. Mechanistically, NSD3S binds to MYC and reduces the association of F-box and WD repeat domain containing 7 (FBXW7) with MYC, which results in suppression of FBXW7-mediated proteasomal degradation of MYC and an in increase MYC protein half-life.

These results support a critical role for NSD3S in the regulation of MYC function and provide a novel mechanism for NSD3S oncogenic function through inhibition of FBXW7-mediated degradation of MYC. The study suggests a novel regulatory axis between NSD3S and MYC and a potential therapeutic approach for treating patients with MYC-driven tumors.

Table of Contents

Chapter 1: Introduction. 1

1.1 Cancer 2

1.2 Protein-Protein Interaction Network. 2

1.3 MYC.. 6

1.3.1 MYC protein structure. 6

1.3.2 MYC alterations in cancer 9

1.3.3 MYC interactome. 10

1.3.4 MYC transcriptional regulation. 13

1.3.5 MYC degradation. 13

1.3.6 Functions of MYC in tumorigenesis. 19

1.3.7 Approaches to target MYC.. 20

1.4 Novel MYC binding partners. 20

1.5 Epigenetics and NSD3. 21

1.5.1 NSD3 protein structure. 22

1.5.2 NSD3 alterations in cancer 26

1.5.3 NSD3 interactome. 27

1.5.4 Function of NSD3 in tumorigenesis. 28

1.6 Scope of the dissertation. 30

Chapter 2: The OncoPPi network of cancer-focused protein-protein interactions to inform novel MYC binding partners. 31

2.1 Introduction. 32

2.2 Material and methods. 33

2.3 Results. 39

2.3.1 Defining the OncoPPi network. 39

2.3.1 Defining MYC as a node for signaling pathway connection. 43

2.3.3 NSD3S is a novel MYC binding partner 47

2.3.4 NSD3S regulates MYC half-life and transcriptional activity. 52

2.3.5 Connection between MYC and BRD4 through NSD3S. 55

2.4 Discussion. 58

Chapter 3: NSD3S stabilizes MYC through hindering its interaction with FBXW7. 61

3.1 Introduction. 62

3.2 Material and Methods. 64

3.3 Results. 69

3.3.1 NSD3S binds MYC at a distinct site. 69

3.3.2 A 15 amino acid peptide of NSD3S mediates MYC binding. 72

3.3.3 NSD3S-pep15 is required for functional regulation of MYC.. 76

3.3.4 NSD3S stabilizes MYC by interfering with FBXW7-mediated proteasomal degradation. 80

3.4 Discussion. 89

Chapter 4: Discussion and future directions. 92

4.1 MYC as a central node in the PPI network. 93

4.2 Elements of MYC and NSD3S relevant for binding. 94

4.3 NSD3S regulates MYC function through a well-defined degradation pathway. 95

4.4 Potential mechanisms for the increase of MYC transcriptional activity by NSD3S. 96

4.5 Therapeutic implications. 97

4.5.1 MYC.. 98

4.5.2 NSD3S. 98

4.6 Summary and future directions. 98

References. 102

Appendix. 124

A.1 Connection between NSD3S/MYC/MAX.. 125

A.2 Ultra-high-throughput screening of small molecules for MYC/NSD3S PPI. 128

A.3 Ultra-high-throughput screening of small molecules for NSD3S/BRD4 PPI. 131

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