Cooperativity between MYC and other oncogenic factors : implications for tumorigenesis and targeting of MYC

Abstract

Normal cell homeostasis in a tissue requires a delicate balance between cell growth, maintenance and death, tightly controlled by an intricate interplay between proto-oncogenes and tumor suppressor genes. When the balance is tipped due to genetic or epigenetic lesions in such genes, diseases such as cancer may arise. MYC transcription factors has been known to regulate up to 15% of mammalian genes involved in diverse intracellular programs, including cell cycle regulation, cell growth, differentiation, apoptosis, and senescence, and is deregulated in many human cancers. MYC recruits different co-factors for activation or repression of transcription, such as MAX or Miz-1, respectively. MYC is tightly regulated at multiple levels, including transcription, posttranslational modification and turnover. Ubiquitylation is one such control, and although ubiquitylation is mainly associated with proteasomal degradation, it has also been shown to be involved in non-proteolytic functions such as DNA replication and repair.

Tumorigenesis is a multistep process that involves activating or inactivating mutations or epigenetic changes in more than one gene to confer growth advantages to the cell. MYC is known to cooperate with another oncoprotein, RAS, to transform rodent cells. While RAS has been found to suppress MYC-induced apoptosis, MYC also inhibits RAS-induced senescence, thereby blocking two main anti-tumorigenic mechanisms in the cell and may, at least in part, explain the basis for the MYC/RAS cooperativity. Inactivation of MYC in mouse tumor models demonstrated tumor regression with welltolerated side effects, suggesting that MYC is a potential and suitable target for anti-cancer therapy. However, pharmacological targeting of transcription factors is considered difficult and no anti-MYC drugs are clinically available today.

In this thesis, we deepen our understanding on MYC biology by studying different proteins that cooperate and interact with MYC (Paper I to III), and identify small molecules that would target specific interactions involving MYC (Paper IV). In Paper I, we found that oncogenic MYC and RAS do not cooperate to cancel out each other’s intrinsic anti-tumorigenic barrier, namely apoptosis and senescence, in normal human fibroblasts as they do in murine fibroblasts, even in the absence of the tumor suppressor p53. This is in contrast to previous results from human melanocytes, where MYC was reported to suppress BRAF- and partially NRAS-induced senescence, thus suggesting that these antitumor barriers are orchestrated differently in different species and in different cell types. In Papers II and III, we discovered new regulatory mechanisms for MYC. In Paper II, we found that the cyclin-dependent kinase (CDK) inhibitor p27KIP1 (p27) binds MYC and targets it for degradation. p27 is upregulated by interferon-ɣ (IFN-ɣ) and by other growth inhibitory signals. We also found that IFN-ɣ treatment leads to the degradation of MYC, mediated by upregulation of p27. There is significant clinical relevance between high activity of nuclear p27 levels and low MYC expression in tumor samples, and this correlates with a good prognosis and a positive clinical outcome. This may provide insights into strategies to target MYC-driven tumors, for example by finding ways to upregulate p27 expression and activity, utilizing IFN-ɣ in treatment modalities, stimulating immune cells to produce IFN-ɣ by immunotherapy and finding methods to combine these strategies to combat MYC-driven tumors.

In Paper III, we uncovered a novel F-box protein, FBXO28, that ubiquitylates MYC in a nonproteolytic manner, and enhances MYC transcriptional activity and downstream pathways. Phosphorylation of SCFFBXO28 by CDK1/2 during the cell cycle is required for its efficient ubiquitylation of MYC. Depletion of FBXO28 or expression of its dominant negative F-box mutant, negates this function and results in reduction of MYC-driven transcription, transformation and tumorigenesis. High MYC expression coupled with high FBXO28 expression and phosphorylation are strong and independent predictors of poor prognosis in human breast cancer. Our data suggests that the CDK-FBXO28-MYC axis is a potential molecular drug target in MYC-driven cancers, including breast cancer. In Paper IV, we conducted a small molecule screen and found, MYCMI-6, that binds MYC, inhibits MYC/MYCN:MAX interactions, and impeded tumor cell growth in a MYCdependent manner in a variety of tumor cell cultures and in a mouse tumor model of MYCNamplified neuroblastoma. Importantly, this compound is highly specific and potent, has a good therapeutic window and does not have severe side effects. This discovery provides proof of principle of protein-protein targeting. MYCMI-6 can be used as a molecular tool to study MYC:MAX interactions and is a good candidate for drug development.

Altogether, the projects involved in this thesis provide insights into molecular pathways involved in MYC oncogenic activity, regulation, and transcription functions, shed light in MYC-RAS cooperativity, identified new proteins interacting with MYC and small molecules interfering with MYC function. This is of importance not only to increase the basic knowledge on mechanisms through which MYC contributes to tumor development, but will hopefully also contribute to the development of new therapeutic strategies to combat MYCdriven cancer in the future.