Validation of MKK3/MYC PPI as a Potential Therapeutic Target Öffentlichkeit
Yang, Xuan (Spring 2019)
Abstract
Oncogenic master transcription factor MYC is amplified in nearly all cancers. However, MYC lacks known enzymatic activity and its targeting by small molecules is highly challenging. Meanwhile, MYC transcription activity is controlled by other proteins through the regulation of its protein level and expression. Previously, mitogen-activated protein kinase kinase 3 (MKK3) has been identified as a novel binding partner of the major tumor driver MYC. The protein-protein interaction (PPI) between MKK3 and MYC leads to upregulation of MYC transcriptional activity in cancer cells. We discovered that ASK1 (MAP3K5) a major activator of MKK3 can serve as a potential regulatory switch between MKK3 pro-inflammatory and pro-apoptotic p38 MAPK activation and oncogenic activation of MYC. GSK3β, a negative regulator that promotes MYC degradation, has also been identified as a novel binding partner of MKK3. We found that inhibition of GSK3β-dependent MYC degradation is a potential mechanism MKK3 uses to enhance MYC stability. These data provide new mechanistic insights into MKK3-dependent MYC activation. To interrogate MKK3/MYC binding interface l developed inhibitory peptides derived from MYC HLH domain. Antitumor effect of the peptides suggests that MKK3/MYC PPI is druggable and its inhibition has potential therapeutic effect. Through the high-throughput screening of compounds with known bioactivity we have discovered first-in-class inhibitors for MKK3/MYC PPI, including the quinoline derivative SGI-1027. Previous studies defined SGI-1027 as a probe for DNA methyltransferase activity, however other biological targets for this molecule remained unexplored. we found that SGI-1027 inhibits MKK3/MYC PPI in multiple assays at low micromolar range, and demonstrates selectivity against other well-defined MYC and MKK3 PPIs, such as MYC/MAX and MKK3/p38 PPIs. Disruption of MKK3/MYC PPI correlates with the suppression of MYC transcriptional activity and reduced proliferation in multiple cancer cell lines. Together, our findings define MKK3/MYC PPI interface as a new promising target to regulate MYC activation. These data build a strong basis for further investigation of MKK3/MYC PPI dependency for therapeutic discovery in cancer.
Table of Contents
Table of Contents
Introduction 1
PPIs as Promising Targets for Therapeutic Discoveries 2
MYC as a Tough Target Associated with Various Diseases 3
Targeting MYC for Therapeutic Development Remains Challenging 4
OncoPPi-informed discovery of MKK3 as a Novel Binding Partner of MYC 5
MKK3/MYC PPI Validations 6
Interrogation of MKK3/MYC PPI Interface 7
Functional Studies Indicate MKK3/MYC PPI as a Potential Therapeutic Target 8
Scope of the Thesis 9
Materials and Methods 12
Reagents 13
Cell Line and Culture Conditions 13
DNA Constructs 14
GST-pull down Assay 14
Western Blot 14
TR-FRET Measurements 15
Development of MKK3/MYC TR-FRET Assay in a 384-Well HTS Format 15
Miniaturization of the Assay into a 1,536-Well uHTS Format 16
Pilot Screening for Potential MKK3/MYC Inhibitors through uHTS in a 1536-Well Format
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Dose-Response TR-FRET Validation for Compound Hits 17
Data analysis 18
MYC reporter assay 19
Computational modeling 19
Results 20
Shorter truncations were designed and generated from MYC bHLH domain 21
MKK3/MYC PPI can be disrupted by inhibitory peptides 22
Overexpression of MKK3/MYC inhibitory peptide correlates with anti-tumor effects 22
Further narrow-down of MKK3-binding site on MYC 25
Development of the ultra-high-throughput screening assay for MKK3/MYC PPI inhibitors 26
The uHTS screening for MKK3/MYC PPI inhibitors 28
Validation of SGI-1027 as the disruptor of MKK3/MYC PPI 30
SGI-1027 shows significant MKK3/MYC PPI selectivity 33
Computational modeling reveals SGI-1027-binding site on MKK3 36
SGI-1027 suppresses MYC transcriptional activity and proliferation of cancer cell lines 37
MKK3 knock-down results in decreased MYC transcriptional activity 39
Suppressive effect of SGI-1027 on MYC activity and cell proliferation correlates with inhibition of MKK3/MYC PPI 39
Identification of potential regulators for MKK3/MYC PPI 42
ASK1 is a potential modulator of MKK3/MYC PPI 42
MKK3 interferes with MYC/GSK3β interaction 45
Discussion 47
Future directions 52
References 53
Table of Figures
Figure 1. Protein-protein interactions emerging as promising targets. 2
Figure 2. Master transcription factor MYC is tightly related to tumorigenesis. 3
Figure 3. Co-crystallization reveals binding characteristics for MYC/MAX PPI. 4
Figure 4. MKK3 (MAP2K3) identified as a major hub in the OncoPPi network. 5
Figure 5. MKK3/MYC PPI was validated in multiple orthogonal assays. 6
Figure 6. Identification of binding-domains on MKK3 and MYC responsible for their interaction 7
Figure 7. MKK3 inhibits MYC degradation and upregulates its activity. 9
Figure 8. Identification of MKK3-binding site on MYC HLH-domain. 21
Figure 9. MYC 370-413 selectively inhibits the interaction between MKK3 and MYC. 23
Figure 10. Overexpression of peptide antagonist correlates with anti-tumor effect in cancer cells.
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Figure 11. MKK3-binding domain on MYC narrowed down to MYC 380-406. 25
Figure 12. Evaluation of the MKK3/MYC PPI TR-FRET assay. 27
Figure 13. Pilot uHTS screening for MKK3/MYC PPI inhibitors. 29
Figure 14. Dose-response validation of primary hits detected in uHTS assay. 31
Figure 15. Orthogonal validations of SGI-1027 as MKK3/MYC PPI inhibitor. 32
Figure 16. SGI-1027 does not disrupt the PPIs between major MKK3 and MYC binding partners.
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Figure 17. SGI-1027 showed selectivity against other MKK3 PPIs. 35
Figure 18. Molecular computational modeling reveals SGI-1 027-binding site on MKK3. 36
Figure 19. SGI-1027 suppresses MYC transcriptional activity and proliferation of cancer cell lines. 38
Figure 20. MKK3 knock-down results in decreased MYC transcriptional activity. 40
Figure 21. Sensitivity of HCT116 cells to SGI-1027 is MKK3 dependent. 41
Figure 22. ASK1 and GSK3β can inhibit MKK3/MYC PPI. 43
Figure 23. ASK1 is verified as a potential modulator of MKK3/MYC PPI. 44
Figure 24. MKK3 promotes MYC stability through inhibiting GSK3β-dependent MYC degradation. 46
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