Establishment of primary mouse embryo fibroblasts (MEFs) as continuously growing cell lines is normally accompanied by loss of the p53 or p19ARF tumor suppressors, which act in a common biochemical pathway. myc rapidly activates ARF and p53 gene expression in primary MEFs and triggers replicative crisis by inducing apoptosis. MEFs that survive myc overexpression sustain p53 mutation or ARF loss during the process of establishment and become immortal. MEFs lacking ARF or p53 exhibit an attenuated apoptotic response to myc ab initio and rapidly give rise to cell lines that proliferate in chemically defined medium lacking serum. Therefore, ARF regulates a p53-dependent checkpoint that safeguards cells against hyperproliferative, oncogenic signals. The INK4a-ARF locus is a common target of deletion and mutation in human cancers, possibly second in frequency only to p53. The product of the INK4a gene, p16 INK4a, acts as an inhibitor of cyclin D-dependent kinases, preventing them from phosphorylating the retinoblastoma (pRb) protein and thus inhibiting S-phase entry during the cell division cycle (Serrano et al. 1993). A second product of this locus, p19 ARF

Transgenic mice expressing the c-Myc oncogene driven by the immunoglobulin heavy chain enhancer (Eµ) develop B-cell lymphoma and exhibit a mean survival time of approximately 6 months. The protracted latent period before the onset of frank disease likely reflects the ability of c-Myc to induce a p53-dependent apoptotic program that initially protects animals against tumor formation but is disabled when overtly malignant cells emerge. In cultured primary mouse embryo fibroblasts, c-Myc activates the p19 ARF -Mdm2-p53 tumor suppressor pathway, enhancing p53-dependent apoptosis but ultimately selecting for surviving immortalized cells that have sustained either p53 mutation or biallelic ARF deletion. Here we report that p53 and ARF also potentiate Myc-induced apoptosis in primary pre-B-cell cultures, and that spontaneous inactivation of the ARF-Mdm2-p53 pathway occurs frequently in tumors arising in Eµ-myc transgenic mice. Many Eµ-myc lymphomas sustained either p53 (28%) or ARF (24%) loss of function, whereas Mdm2 levels were elevated in others. Its overexpression in some tumors lacking p53 function raises the possibility that Mdm2 can contribute to lymphomagenesis by interacting with other targets. Eµ-myc transgenic mice hemizygous for ARF displayed accelerated disease (11-week mean survival), and 80% of these tumors lost the wild-type ARF allele. All ARF-null Eµ-myc mice died of lymphoma within a few weeks of birth. About half of the tumors arising in ARF hemizygous or ARF nullizygous Eµ-myc transgenic mice also overexpressed Mdm2. Therefore, Myc activation strongly selects for spontaneous inactivation of the ARF-Mdm2-p53 pathway in vivo, canceling its protective checkpoint function and accelerating progression to malignancy.

SUMMARY MYC is an oncoprotein transcription factor that is overexpressed in the majority of malignancies. The oncogenic potential of MYC stems from its ability to bind regulatory sequences in thousands of target genes, which depends on interaction of MYC with its obligate partner, MAX. Here, we show that broad association of MYC with chromatin also depends on interaction with the WD40-repeat protein WDR5. MYC binds WDR5 via an evolutionarily conserved “MYC box IIIb” motif that engages a shallow, hydrophobic, cleft on the surface of WDR5. Structure-guided mutations in MYC that disrupt interaction with WDR5 attenuate binding of MYC to ~80% of its chromosomal locations and disable its ability to promote induced pluripotent stem cell formation and drive tumorigenesis. Our data reveal WDR5 as a key determinant for MYC recruitment to chromatin and uncover a tractable target for the discovery of anti-cancer therapies against MYC-driven tumors.

Key Points High-throughput sequencing of MF revealed multiple mutations within epigenetic and cytokine pathways that may drive disease. Pharmacologically targeting the JAK3 pathway in MF results in cell death and may be an effective treatment of this disease.

Myc requires de novo protein synthesis, indicating that repression is indirect. Importantly, the suppression of Bcl-2 or Bcl-X L by Myc is corrupted during Myc-induced tumorigenesis, as Bcl-2 and/or Bcl-X L levels are markedly elevated in over one-half of all lymphomas arising in E-myc transgenic mice. Bcl-2 and/or Bcl-X L overexpression did not correlate with loss of ARF or p53 function in tumor cells, indicating that these two apoptotic pathways are inactivated independently. Therefore, the suppression of Bcl-X L or Bcl-2 expression represents a physiological Myc-induced apoptotic pathway that is frequently bypassed during lymphomagenesis.