MYC and RAS are unable to cooperate in overcoming cellular senescence and apoptosis in normal human fibroblasts.

The MYC and RAS oncogenes are sufficient for transformation of normal rodent cells. This cooperativity is at least in part based on suppression of RAS-induced cellular senescence by MYC and block of MYC-induced apoptosis by RAS - thereby canceling out two main barriers against tumor development. However, it remains unclear whether MYC and RAS cooperate in this way in human cells, where MYC and RAS are not sufficient for transformation. To address this question, we established a combined Tet-inducible H-RASV12 and hydroxytamoxifen-inducible MycER system in normal human BJ fibroblasts. We show here that activation of RAS alone induced senescence while activation of MYC alone or together with RAS triggered DNA damage, induction of p53 and massive apoptosis, suggesting that RAS cannot rescue MYC-induced apoptosis in this system. Although coexpression with MYC reduced certain RAS-induced senescence markers (histone H3 lysine 9 trimethylation and senescence-associated ß-GAL activity), the induction of the senescence marker p16INK4A was further enhanced and the culture ceased to proliferate within a few days, revealing that MYC could not fully suppress RAS-induced senescence. Furthermore, depletion of p53, which enhanced proliferation and rescued the cells from RAS-induced senescence, did not abrogate MYC-induced apoptosis. We conclude that MYC and RAS are unable to cooperate in overcoming senescence and apoptosis in normal human fibroblasts even after depletion of p53, indicating that additional oncogenic events are required to abrogate these fail-safe mechanisms and pave the way for cellular transformation. These findings have implications for our understanding of the transformation process in human cells. Abbreviations and acronyms: CDK: Cyclin-dependent kinase; DDR: DNA damage response; DOX: Doxycycline; EdU: 5-ethynyl-2'-deoxyuridine; FACS: Fluorescence Activated Cell Sorting; MycER: MYC-estrogen receptor; OHT: 4-hydroxytamoxifen; OIS: Oncogene-induced senescence; PP2A: Protein phosphatase 2A; ROS: Reactive oxygen species; SA-ß-GAL: Senescence-associated ß-galactosidase; SAHF: Senescence-associated heterochromatin foci; shRNA: Short hairpin RNA; YFP: Yellow fluorescent protein.

A selective high affinity MYC-binding compound inhibits MYC:MAX interaction and MYC-dependent tumor cell proliferation.

MYC is a key player in tumor development, but unfortunately no specific MYC-targeting drugs are clinically available. MYC is strictly dependent on heterodimerization with MAX for transcription activation. Aiming at targeting this interaction, we identified MYCMI-6 in a cell-based protein interaction screen for small inhibitory molecules. MYCMI-6 exhibits strong selective inhibition of MYC:MAX interaction in cells and in vitro at single-digit micromolar concentrations, as validated by split Gaussia luciferase, in situ proximity ligation, microscale thermophoresis and surface plasmon resonance (SPR) assays. Further, MYCMI-6 blocks MYC-driven transcription and binds selectively to the MYC bHLHZip domain with a KD of 1.6 ± 0.5 µM as demonstrated by SPR. MYCMI-6 inhibits tumor cell growth in a MYC-dependent manner with IC50 concentrations as low as 0.5 µM, while sparing normal cells. The response to MYCMI-6 correlates with MYC expression based on data from 60 human tumor cell lines and is abrogated by MYC depletion. Further, it inhibits MYC:MAX interaction, reduces proliferation and induces massive apoptosis in tumor tissue from a MYC-driven xenograft tumor model without severe side effects. Since MYCMI-6 does not affect MYC expression, it is a unique molecular tool to specifically target MYC:MAX pharmacologically and it has good potential for drug development.

Interferon-γ-induced p27KIP1 binds to and targets MYC for proteasome-mediated degradation.

The Myc oncoprotein is tightly regulated at multiple levels including ubiquitin-mediated protein turnover. We recently demonstrated that inhibition of Cdk2-mediated phosphorylation of Myc at Ser-62 pharmacologically or through interferon (IFN)-γ-induced expression of p27(Kip1) (p27) repressed Myc's activity to suppress cellular senescence and differentiation. In this study we identified an additional activity of p27 to interfere with Myc independent of Ser-62 phosphorylation. p27 is required and sufficient for IFN-γ-induced turnover of Myc. p27 interacted with Myc in the nucleus involving the C-termini of the two proteins, including Myc box 4 of Myc. The C-terminus but not the Cdk2 binding fragment of p27 was sufficient for inducing Myc degradation. Protein expression data of The Cancer Genome Atlas breast invasive carcinoma set revealed significantly lower Myc protein levels in tumors with highly expressed p27 lacking phosphorylation at Thr-157--a marker for active p27 localized in the nucleus. Further, these conditions correlated with favorable tumor stage and patient outcome. This novel regulation of Myc by IFN-γ/p27(KIP1) potentially offers new possibilities for therapeutic intervention in tumors with deregulated Myc.

CDK-mediated activation of the SCF(FBXO) (28) ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer.

SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCF(FBXO28) activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCF(FBXO28) plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer.