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XLSX file, 14K, Gene expression changes common to wild type MYC and all phosphorylation mutants (112 genes).

XLS file, 160K, GO Analysis of significant genes expression changes.

XLS file, 403K, Expression comparison between wild-type MYC and MYC phosphorylation mutants (from Venn diagram)

XLS file, 2439K, MYC ChIP-on-chip data in MCF10A cells.

XLS file, 14K, Phosphopeptide analysis for ectopic MYC in 293Tv cells.

XLS file, 98K, GO analysis of common gene expression changes.

PDF file, 136K.

XLS file, 319K, Expression array analysis of MCF10A 3D cultures at 4 days.

XLS file, 3148K, Compiled expression array and ChIP-on-chip data.

XLSX file, 16K, Gene expression changes common to all gain-of-function MYC phosphorylation mutants (158 genes).

PDF file, 498K, Supplementary Figures S1-6 Supplementary Figure S1: Phosphorylation mutants increase Myc-induced transformation in MCF10A cells. Supplementary Figure S2: Phosphorylation mutants increase Myc-induced transformation in SH-EP cells. Supplementary Figure S3: Phosphorylation mutants do not effect MYC protein stability. Supplementary Figure S4: Serum starvation of MCF10A-GFP and MCF10A-MYC cells. Supplementary Figure S5: MYC protein expression through morphogenesis of MCF10A cells in 3D culture. Supplementary Figure S6: Characterization of day 4 acini for mRNA expression array analysis.

MYC is a key driver of cellular transformation and is deregulated in most human cancers. Studies of MYC and its interactors have provided mechanistic insight into its role as a regulator of gene transcription. MYC has been previously linked to chromatin regulation through its interaction with INI1 (SMARCB1/hSNF5/BAF47), a core member of the SWI/SNF chromatin remodeling complex. INI1 is a potent tumor suppressor that is inactivated in several types of cancers, most prominently as the hallmark alteration in pediatric malignant rhabdoid tumors. However, the molecular and functional interaction of MYC and INI1 remains unclear. Here, we characterize the MYC-INI1 interaction in mammalian cells, mapping their minimal binding domains to functionally significant regions of MYC (leucine zipper) and INI1 (repeat motifs), and demonstrating that the interaction does not interfere with MYC-MAX interaction. Protein-protein interaction network analysis expands the MYC-INI1 interaction to the SWI/SNF complex and a larger network of chromatin regulatory complexes. Genome-wide analysis reveals that the DNA-binding regions and target genes of INI1 significantly overlap with those of MYC. In an INI1-deficient rhabdoid tumor system, we observe that with re-expression of INI1, MYC and INI1 bind to common target genes and have opposing effects on gene expression. Functionally, INI1 re-expression suppresses cell proliferation and MYC-potentiated transformation. Our findings thus establish the antagonistic roles of the INI1 and MYC transcriptional regulators in mediating cellular and oncogenic functions.

The c-MYC (MYC) oncoprotein is deregulated in over 50% of cancers, yet regulatory mechanisms controlling MYC remain unclear. To this end, we interrogated the MYC interactome using BioID mass spectrometry (MS) and identified PP1 (protein phosphatase 1) and its regulatory subunit PNUTS (protein phosphatase-1 nuclear-targeting subunit) as MYC interactors. We demonstrate that endogenous MYC and PNUTS interact across multiple cell types and that they co-occupy MYC target gene promoters. Inhibiting PP1 by RNAi or pharmacological inhibition results in MYC hyperphosphorylation at multiple serine and threonine residues, leading to a decrease in MYC protein levels due to proteasomal degradation through the canonical SCFFBXW7 pathway. MYC hyperphosphorylation can be rescued specifically with exogenous PP1, but not other phosphatases. Hyperphosphorylated MYC retained interaction with its transcriptional partner MAX, but binding to chromatin is significantly compromised. Our work demonstrates that PP1/PNUTS stabilizes chromatin-bound MYC in proliferating cells., Deregulated MYC activity is oncogenic and is deregulated in a large fraction of human cancers. Here the authors find that protein phosphatase 1 and its regulatory subunit PNUTS controls MYC stability and its interaction with chromatin.

We determined that the small, ubiquitin-related modifier–specific isopeptidase, SENP2, is dynamically associated with nuclear pore complexes (NPCs). This association is determined by the activities of three N-terminal signals in SENP2: a high-affinity nuclear localization sequence, an Nup107-160–binding element, and a nuclear export signal. NPC association, and its potential regulation, affects SENP2 accessibility to substrates., The association of small, ubiquitin-related modifier–specific isopeptidases (also known as sentrin-specific proteases, or SENPs) with nuclear pore complexes (NPCs) is conserved in eukaryotic organisms ranging from yeast to mammals. However, the functional significance of this association remains poorly understood, particularly in mammalian cells. In this study, we have characterized the molecular basis for interactions between SENP2 and NPCs in human cells. Using fluorescence recovery after photobleaching, we demonstrate that SENP2, although concentrated at the nuclear basket, is dynamically associated with NPCs. This association is mediated by multiple targeting elements within the N-terminus of SENP2 that function cooperatively to mediate NPC localization. One of these elements consists of a high-affinity nuclear localization signal that mediates indirect tethering to FG-repeat–containing nucleoporins through karyopherins. A second element mediates interactions with the Nup107-160 nucleoporin subcomplex. A third element consists of a nuclear export signal. Collectively, our findings reveal that SENP2 is tethered to NPCs through a complex interplay of interactions with nuclear import and export receptors and nucleoporins. Disruption of these interactions enhances SENP2 substrate accessibility, suggesting an important regulatory node in the SUMO pathway.

BioID was performed using FlagBirA⁎ (the R118G biotin ligase mutant protein) and FlagBirA⁎-Myc in HEK293 T-REx cells maintained both under standard cell culture conditions and as mouse xenografts. The mass spectrometry dataset acquired in this study has been uploaded to the MassIVE repository with ID: MSV000078518, and consists of 28 ⁎.raw MS files acquired on an Orbitrap Velos instrument, collected in data-dependent mode. iProphet processed MS/MS search results are also included as a reference. This study has been published as “BioID identifies novel c-MYC interacting partners in cultured cells and xenograft tumors”, by Dingar et al. in the Journal of Proteomics, 2014 [1].

Transforming members of the MYC family (MYC, MYCL1, and MYCN) encode transcription factors containing six highly conserved regions, termed MYC homology boxes (MBs). By conducting proteomic profiling of the MB interactomes, we demonstrate that half of the MYC interactors require one or more MBs for binding. Comprehensive phenotypic analyses reveal that two MBs, MB0 and MBII, are universally required for transformation. MBII mediates interactions with acetyltransferase-containing complexes, enabling histone acetylation, and is essential for MYC-dependent tumor initiation. By contrast, MB0 mediates interactions with transcription elongation factors via direct binding to the general transcription factor TFIIF. MB0 is dispensable for tumor initiation but is a major accelerator of tumor growth. Notably, the full transforming activity of MYC can be restored by co-expression of the non-transforming MB0 and MBII deletion proteins, indicating that these two regions confer separate molecular functions, both of which are required for oncogenic MYC activity.

The c-MYC transcription factor is a master regulator of many cellular processes and deregulation of this oncogene has been linked to more than 50% of all cancers. This deregulation can take many forms, including altered post-translational regulation. Here, using immunoprecipitation combined with mass spectrometry, we identified a MYC SUMOylation site (K326). Abrogation of signaling through this residue by substitution with arginine (K326R) has no obvious effects on MYC half-life, intracellular localization, transcriptional targets, nor on the biological effects of MYC overexpression in two different cell systems assessed for soft agar colony formation, proliferation, and apoptosis. While we have definitively demonstrated that MYC SUMOylation can occur on K326, future work will be needed to elucidate the mechanisms and biological significance of MYC regulation by SUMOylation.

In this paper, we apply the fuzzy linear regression (FLR) with fuzzy intervals analysis into a neural network classification model. The FLR works as a data handler and separates the data sample into two groups. By training two independent neural works with these two groups, we can better describe the distribution space of the corresponding data sample with two different functions, rather than using only one function. The experimental result shows that our approach improves the accuracy of classification.

Despite its central role in human cancer, MYC deregulation is insufficient by itself to transform cells. Because inherent mechanisms of neoplastic control prevent precancerous lesions from becoming fully malignant, identifying transforming alleles of MYC that bypass such controls may provide fundamental insights into tumorigenesis. To date, the only activated allele of MYC known is T58A, the study of which led to identification of the tumor suppressor FBXW7 and its regulator USP28 as a novel therapeutic target. In this study, we screened a panel of MYC phosphorylation mutants for their ability to promote anchorage-independent colony growth of human MCF10A mammary epithelial cells, identifying S71A/S81A and T343A/S344A/S347A/S348A as more potent oncogenic mutants compared with wild-type (WT) MYC. The increased cell-transforming activity of these mutants was confirmed in SH-EP neuroblastoma cells and in three-dimensional MCF10A acini. Mechanistic investigations initiated by a genome-wide mRNA expression analysis of MCF10A acini identified 158 genes regulated by the mutant MYC alleles, compared with only 112 genes regulated by both WT and mutant alleles. Transcriptional gain-of-function was a common feature of the mutant alleles, with many additional genes uniquely dysregulated by individual mutant. Our work identifies novel sites of negative regulation in MYC and thus new sites for its therapeutic attack. Cancer Res; 73(21); 6504–15. ©2013 AACR.

Modifying Deubiquitinases Protein ubiquitination is a widespread mechanism for cellular regulation, and new regulators are valuable research tools and may help to generate therapeutic small molecules. Ernst et al. (p. 590 , published online 3 January) used known crystal structures to roughly define the interaction domain between a ubiquitin-specific protease and a ubiquitinated substrate and then screened ubiquitin variants with changes in these residues to find variants that acted as potent and specific regulators that could modify ubiquitin pathway regulation in cells.

Human cytomegalovirus infections involve the extensive modification of host cell pathways, including cell cycle control, the regulation of the DNA damage response, and averting promyelocytic leukemia (PML)-mediated antiviral responses. The UL35 gene from human cytomegalovirus is important for viral gene expression and efficient replication and encodes two proteins, UL35 and UL35a, whose mechanism of action is not well understood. Here, affinity purification coupled with mass spectrometry was used to identify previously unknown human cellular targets of UL35 and UL35a. We demonstrate that both viral proteins interact with the ubiquitin-specific protease USP7, and that UL35 expression can alter USP7 subcellular localization. In addition, UL35 (but not UL35a) was found to associate with three components of the Cul4 DCAF1 E3 ubiquitin ligase complex (DCAF1, DDB1, and DDA1) previously shown to be targeted by the HIV-1 Vpr protein. The coimmunoprecipitation and immunofluorescence microscopy of DCAF1 mutants revealed that the C-terminal region of DCAF1 is required for association with UL35 and mediates the dramatic relocalization of DCAF1 to UL35 nuclear bodies, which also contain conjugated ubiquitin. As previously reported for the Vpr-DCAF1 interaction, UL35 (but not UL35a) expression resulted in the accumulation of cells in the G 2 phase of the cell cycle, which is typical of a DNA damage response, and activated the G 2 checkpoint in a DCAF1-dependent manner. In addition, UL35 (but not UL35a) induced γ-H2AX and 53BP1 foci, indicating the activation of DNA damage and repair responses. Therefore, the identified interactions suggest that UL35 can contribute to viral replication through the manipulation of host responses.

The MYC oncogene is not only deregulated in cancer through abnormally high levels of expression, but also through oncogenic lesions in upstream signalling cascades. Modelling MYC deregulation using signalling mutants is a productive research strategy. For example, the MYC threonine-58 to alanine substitution mutant (T58A) within MYC-homology box 1 is more transforming than wild-type (WT) MYC, because of decreased apoptosis and increased protein stability. Understanding the regulatory mechanisms controlling T58 phosphorylation has led to new approaches for the development of MYC inhibitors. In this manuscript, we have extensively characterized a MYC signalling mutant in which six lysine residues near the highly conserved MYC homology box IV and basic region have been substituted to arginines (6KR). Previous literature suggests these lysines can undergo both ubiquitylation and acetylation. We show MYC 6KR is able to fully rescue the slow growth phenotype of HO15.19 MYC-null fibroblasts, and promote cell cycle entry of serum-starved MCF10A cells. Remarkably, 6KR increased anchorage-independent colony growth compared with WT MYC in both SH-EP and MCF10A cells. Moreover, it was also more potent in promoting xenograft tumour growth of Rat1A and SH-EP cells. Combined, our data identify this region and these six lysines as important residues for the negative regulation of MYC-induced transformation. Mechanistically, we demonstrate that, unlike T58A, the increased transformation is not a result of increased protein stability or a reduced capacity for 6KR to induce apoptosis. Through expression analysis and luciferase reporter assays, we show that 6KR has increased transcriptional activity compared with WT MYC. Combined, through a comprehensive evaluation across multiple cell types, we identify an important regulatory region within MYC. A better understanding of the full scope of signalling through these residues will provide further insights into the mechanisms contributing to MYC-induced tumorigenesis and may unveil novel therapeutic strategies to target Myc in cancer.

The c-MYC transcription factor is a master regulator of many cellular processes and deregulation of this oncogene has been linked to more than 50% of all cancers. In normal cells, MYC is tightly controlled at a number of steps, including at the transcriptional, translational and post-translational levels. Altered regulation at any of these steps can result in deregulated, oncogenic MYC. One well-studied canonical pathway that is known to regulate MYC activity and stability at the post-translational level is the GSK3 pathway. The GSK3-FBXW7 axis regulates MYC via phosphorylation at T58, followed by ubiquitylation of MYC by the E3 ubiquitin ligase complex SCF-FBXW7 and subsequent proteasomal degradation. Accordingly, substituting threonine 58 with alanine (T58A) confers increased stability and transformative potential. Thus, characterizing the post-translational modifications (PTMs) of MYC can lead to a better understanding of the regulatory mechanisms controlling this potent oncogene. SUMOylation is a post-translational modification that utilizes a series of E1, E2 and E3 proteins for conjugation of a small ubiquitin-like modifier (SUMO) moiety to its target protein. Growing evidence indicates that SUMOylation has many important roles in the cell, such as response to cellular stressors and transcriptional regulation. Moreover, recent reports have unveiled a potential role for SUMOylation in MYC-driven tumourigenesis. Here, using immunoprecipitation combined with mass spectrometry, we identified a MYC SUMOylation site (K326). Abrogation of signaling through this residue by substitution with arginine (K326R) has no obvious effects on MYC half-life, intracellular localization, transcriptional targets, nor on the biological effects of MYC overexpression in three different cell systems assessed for soft agar colony formation, proliferation, and apoptosis. While we have definitively demonstrated that MYC SUMOylation can occur on K326, future work will be needed to elucidate the mechanisms and biological significance of MYC regulation by SUMOylation. Citation Format: Manpreet Kalkat, Pak-Kei Chan, Amanda R. Wasylishen, Tharan Srikumar, Sam S. Kim, Romina Ponzielli, David P. Bazett-Jones, Brian Raught, Linda Z. Penn. Identification of c-MYC SUMOylation by mass spectrometry. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A08.

The c-MYC (MYC) oncogene plays an important role in tumorigenesis and is implicated in >50% of all human cancers. Deregulation of MYC can occur through abnormally high expression levels, but also through oncogenic lesions in upstream signaling cascades. The study of these signaling pathways have provided an alternative approach for the development of MYC-targeted therapeutics. For example, the study of post-translational modifications (PTMs) of MYC, such as P-T58 and the T58A gain-of-function mutant, identified FBXW7 as a tumor suppressor and the deubiquitinating enzyme USP28 as a therapeutic target. We considered that MYC is highly modified post-translationally and that unknown mechanistic pathways may be modifying residues, in addition to T58, in order to control MYC stability and/or function. These undiscovered pathways may therefore provide additional opportunities for the development of MYC-targeted therapeutics. These considerations led to recent work in the Penn lab that uncovered clusters of negatively regulating residues of MYC function. These residues include S71/S81, a cluster of residues referred to as MYC-4 (T343, S344, S347 and S348) and a cluster of 6 lysine residues (6K) at the C-terminal end of MYC (K298, K317, K323, K326, K341 and K355). These negatively regulating residues were characterized using alanine (S71/S81 and 340 cluster) and arginine (C-terminal lysines) substitution mutants in our established transformation assays. The S71/S81A and MYC-4A mutants scored with having gain-of-function activity in comparison to wild-type MYC in multiple transformation assays including growth in soft agar and the disruption of regular acini formation using a normal, immortalized MCF10A cell line. In addition, these mutants were shown to regulate additional genes compared to wild-type MYC using genome-wide mRNA expression analysis of MCF10A acini, suggesting that these MYC proteins have gained additional transcriptional targets. Additionally, substitution of the C-terminal lysine residues with arginine (6KR) also revealed gain-of-function activity. 6KR expressing MCF10A and SH-EP cells had increased anchorage-independent growth compared to cells expressing wild-type MYC and was also more potent in promoting xenograft tumor growth of Rat1A and SH-EP cells. Interestingly, all three mutants do not have extended half-lives as seen with T58A, suggesting that functional activity and not stability is contributing to these transformative phenotypes. The above mutants reveal that each of S71/S81, MYC-4 and C-terminal 6K residues are critically important for the negative regulation of MYC-induced transformation. To further explore these regions of MYC, we used mass spectrometry to identify post-translational modifications that occurred on MYC in growing cells. These data confirm phosphorylation events on S71/81 as well as at MYC-4A. Strikingly, three modifications were directly observed on three of the six lysine residues; acetylation of lysine 323, ubiquitylation of lysine 355 and SUMOylation of lysine 326. The importance of these modifications and the roles that these modifications have in regulating MYC activity are currently under investigation using our established transformation assays. I now aim to understand the contribution of single or multiple modifications within the indicated clusters and how these modifications modulate MYC activity. Citation Format: Corey Lourenco, Amanda Wasylishen, Michelle Chan-Seng-Yue, Christina Bros, Dharmendra Dingar, William Tu, Manpreet Kalkat, Pak-Kei Chan, Peter Mullen, Brian Raught, Paul Boutros, Linda Penn. The myc post-translational landscape: How novel gain-of-function mutants are revealing new stability and functional regulatory systems. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A10.

Myc oncoprotein is a major driver of cancer initiation and progression, and thus targeting its activity would mark a key therapeutic advance. In a genetic preclinical mouse model, systemic Myc inhibition using the dominant-negative Myc mutant, termed Omomyc, showed that Ras-driven lung cancer could be eradicated without any harmful long-term effects to the animal. However, developing an anti-cancer agent that directly binds and inhibits Myc has not been possible, to date. Therefore, new strategies are required to inhibit Myc in cancer. Understanding the Myc interactome may unravel novel approaches to target Myc in cancer. The BioID proximity-based biotin labeling technique was recently developed for the characterization of protein-protein interaction networks. In BioID, the protein of interest is expressed as a fusion partner biotin ligase (BirA*), which activates biotin. The active biotin reacts with lysine residues on nearby polypeptides. Following a stringent cell lysis and streptavidin-sepharose pulldown, biotinylated proteins can be identified using MS. To date, this method has been applied to a number of different polypeptides expressed in cultured cells. Here we report the adaptation of BioID to the identification of protein-protein interactions surrounding the Myc oncoprotein in human cells grown both under standard culture conditions and in mice as tumor xenografts. Notably, in vivo BioID yielded >100 high confidence Myc interacting proteins, including >30 known binding partners such as MAX (Myc-associated factor X), TRRAP (transformation/transcription domain-associated protein), the enhancer of polycomb homologs 1 and 2 (EPC1, EPC2), lysine acetyltransferase 5 (KAT5). Putative novel Myc interactors include components of the STAGA/KAT5 and SWI/SNF chromatin remodelling complexes (see Penn lab abstract Tu et al), DNA repair and replication factors, general transcription and elongation factors, and transcriptional co-regulators such as the DNA helicase chromodomain 8 (CHD8). Providing additional confidence in these findings, ENCODE ChIP-seq datasets highlight significant coincident binding throughout the genome for the Myc interactors identified here, and we validate the previously unreported CHD8 (an ATP-dependent helicase)-Myc interaction using both a yeast two hybrid analysis and the proximity-based ligation assay (PLA). Additionally, we also validate Myc-BRD4 and Myc-TRIM24 interaction by PLA. In sum, here we identify bona fide interacting partners of Myc in vivo by use of BioID. Our study shows for the first time Myc interactome in vivo, understanding these interactors will shed more light on Myc oncogenesis, which can be used to therapeutically target Myc in cancer. Citation Format: Dharmendra Dingar, Manpreet Kalkat, Pak-Kei Chan, Swneke D. Bailey, Tharan Srikumar, William B. Tu, Etienne Coyaud, Romina Ponzielli, Max Kolyar, Igor Jurisica, Annie Huang, Mathieu Lupien, Brian Raught, Linda Z. Penn. In vivo BioID identifies novel Myc interacting partners. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B04.

The MYC oncogene is a key driver of cellular transformation that is deregulated in more than half of all human cancers. The transcriptional regulatory function of MYC and the wide spectrum of biological processes it mediates are critically tied to its chromatin interactors and epigenetic context. MYC is linked to chromatin remodeling through its interaction with INI1 (SMARCB1/hSNF5/BAF47), a core member of the SWI/SNF complex and a potent tumour suppressor. Recent sequencing efforts in many cancer types also identified frequent mutations in other members of this complex. However, the mechanistic understanding of the SWI/SNF complex in contributing to oncogenesis and its functional and molecular interaction with MYC remain unclear. Herein, we provide a comprehensive characterization of the MYC-INI1 interaction. We demonstrate their direct interaction and extensively delineate their minimal regions of interaction, corresponding to functionally important regions of MYC (leucine zipper) and INI1 (Repeats I and II). Genome-wide analysis reveals that INI1 and other SWI/SNF complex members share significant portions of MYC DNA-binding regions and target genes. Network analysis demonstrates MYC interaction with the SWI/SNF complex and an extended network of shared interactors belonging to additional chromatin regulatory complexes. Collectively, our findings provide significant insight into the interaction of MYC and INI1 at the level of protein-protein and protein-chromatin interactions. Citation Format: William B. Tu, Angelina Stojanova, Romina Ponzielli, Max Kotlyar, Pak-Kei Chan, Paul C. Boutros, Fereshteh Khosravi, Igor Jurisica, Brian Raught, Linda Z. Penn. Interaction of the MYC oncoprotein with the tumor suppressive SWI/SNF complex member INI1. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A36.