Michael Wanzel

Negative Regulation of the Mammalian UV Response by Myc through Association with Miz-1.

The Myc oncoprotein represses initiator-dependent transcription through the POZ domain transcription factor Miz-1. We now show that transactivation by Miz-1 is negatively regulated by association with topoisomerase II binding protein (TopBP1); UV irradiation downregulates expression of TopBP1 and releases Miz-1. Miz-1 binds to the p21Cip1 core promoter in vivo and is required for upregulation of p21Cip1 upon UV irradiation. Using both c-myc(-/-) cells and a point mutant of Myc that is deficient in Miz-1 dependent repression, we show that Myc negatively regulates transcription of p21Cip1 upon UV irradiation and facilitates recovery from UV-induced cell cycle arrest through binding to Miz-1. Our data implicate Miz-1 in a pathway that regulates cell proliferation in response to UV irradiation.

The ubiquitin-specific protease USP28 is required for MYC stability

The MYC proto-oncogene encodes a transcription factor that has been implicated in the genesis of many human tumours. Here, we used a bar-code short hairpin RNA (shRNA) screen to identify multiple genes that are required for MYC function. One of these genes encodes USP28, an ubiquitin-specific protease. USP28 is required for MYC stability in human tumour cells. USP28 binds to MYC through an interaction with FBW7α, an F-box protein that is part of an SCF-type ubiquitin ligase. Therefore, it stabilizes MYC in the nucleus, but not in the nucleolus, where MYC is degraded by FBW7γ. High expression levels of USP28 are found in colon and breast carcinomas, and stabilization of MYC by USP28 is essential for tumour-cell proliferation.

Compassionate use of sorafenib in FLT3-ITD-positive acute myeloid leukemia: sustained regression before and after allogeneic stem cell transplantation.

Acute myeloid leukemia (AML) patients with internal tandem duplication (ITD) mutations in the Fms-like tyrosine-3 (FLT3) gene have a dismal prognosis. Here we report compassionate-use results with the multikinase and FLT3-ITD inhibitor sorafenib for the treatment of relapsed or refractory FLT3-ITD-positive AML. Sorafenib induced clinically meaningful and very rapid responses in all 6 patients treated either before (n = 2), after (n = 3), or both before and after (n = 1) allogeneic stem cell transplantation (allo-SCT). Sorafenib-induced remissions facilitated allo-SCT in 2 of the 3 refractory patients. Two of the 4 patients who were treated after allo-SCT survived 216 and 221 days, respectively, whereas the other 2 remain in ongoing complete molecular remission. Sorafenib response was associated with an inhibition of the antiapoptotic FLT3-ITD target Stat-5 in vivo. Together, sorafenib monotherapy before or after allo-SCT has remarkable clinical activity in poor risk FLT3-ITD-positive AML and deserves further evaluation in prospective clinical trials.

Transcriptional repression by Myc.

The Myc oncoprotein is a transcription factor that can both activate and repress genes. Transcriptional activation by Myc is well understood, but, by contrast, the mechanisms through which Myc represses transcription have remained elusive. Recent evidence suggests that complex formation by Myc with a zinc-finger transcription factor, Miz-1, plays an important role in mediating repression by Myc. The findings might explain how Myc interferes with cell-cycle arrest in response to TGF-beta, APC and DNA damage.

A ribosomal protein L23-nucleophosmin circuit coordinates Miz1 function with cell growth

The Myc-associated zinc-finger protein, Miz1, is a negative regulator of cell proliferation and induces expression of the cell-cycle inhibitors p15Ink4b and p21Cip1. Here we identify the ribosomal protein L23 as a negative regulator of Miz1-dependent transactivation. L23 exerts this function by retaining nucleophosmin, an essential co-activator of Miz1 required for Miz1-induced cell-cycle arrest, in the nucleolus. Mutant forms of nucleophosmin found in acute myeloid leukaemia fail to co-activate Miz1 and re-localize it to the cytosol. As L23 is encoded by a direct target gene of Myc, this regulatory circuit may provide a feedback mechanism that links translation of Myc target genes and cell growth to Miz1-dependent cell-cycle arrest.

Akt and 14-3-3η regulate Miz1 to control cell-cycle arrest after DNA damage

The transcription factor Miz1 is required for DNA-damage-induced cell-cycle arrest. We have now identified 14-3-3η as a gene that inhibits Miz1 function through interaction with its DNA binding domain. Binding of 14-3-3η to Miz1 depends on phosphorylation by Akt and regulates the recovery of cells from arrest after DNA damage. Miz1 has two functions in response to DNA damage: first, it is required for upregulation of a large group of genes, a function that is regulated by c-Myc, but not by 14-3-3η; second, Miz1 represses the expression of many genes in response to DNA damage in an Akt- and 14-3-3η-regulated manner.

p53 DNA Binding Cooperativity Is Essential for Apoptosis and Tumor Suppression In Vivo

Four molecules of the tumor suppressor p53 assemble to cooperatively bind proapoptotic target genes. The structural basis for cooperativity consists of interactions between adjacent DNA binding domains. Mutations at the interaction interface that compromise cooperativity were identified in cancer patients, suggesting a requirement of cooperativity for tumor suppression. We report on an analysis of cooperativity mutant p53E177R mice. Apoptotic functions of p53 triggered by DNA damage and oncogenes were abolished in these mice, whereas functions in cell-cycle control, senescence, metabolism, and antioxidant defense were retained and were sufficient to suppress development of spontaneous T cell lymphoma. Cooperativity mutant mice are nevertheless highly cancer prone and susceptible to different oncogene-induced tumors. Our data underscore the relevance of DNA binding cooperativity for p53-dependent apoptosis and tumor suppression and highlight cooperativity mutations as a class of p53 mutations that result in a selective loss of apoptotic functions due to an altered quaternary structure of the p53 tetramer.

The Arf tumor suppressor protein inhibits Miz1 to suppress cell adhesion and induce apoptosis

Arf assembles a complex containing Miz1, heterochromatin, and histone H3K3 to block expression of genes involved in cell adhesion and signal transduction. The resulting blockade of cell–cell and cell–matrix interactions facilitates elimination of cells carrying oncogenic mutations.

Expression of P27KIP1 is prognostic and independent of MYCN amplification in human neuroblastoma

Amplification of the MYCN gene is significantly associated with an unfavorable prognosis and rapid progression in human neuroblastoma tumors. One potential mechanism by which MYCN may cause these effects is by deregulating cell proliferation. Tissue culture experiments support a model in which MYC genes stimulate cell cycle progression by antagonizing the function of the cell cycle inhibitor p27kip1. In culture, activation of MYC induces both sequestration of p27kip1 by cyclin D complexes and its subsequent proteolytic degradation. We have tested whether this model applies to human neuroblastoma in a retrospective study of 100 primary tumor biopsy samples from neuroblastoma patients with a documented follow‐up. Consistent with this hypothesis, MYCN‐amplified tumors express high levels of both cyclin A and proliferating cell nuclear antigen, 2 marker proteins of cell proliferation. Further, expression levels of p27kip1 are of prognostic significance in human neuroblastoma patients. Similar to tissue culture systems, p27kip1 is sequestered by cyclin D complexes in a subset of human neuroblastoma samples. Surprisingly, however, expression levels of p27kip1 are prognostic independent of MYCN amplification, and tumors that have an amplified MYCN gene do not express elevated levels of D‐type cyclins or contain significantly lower levels of p27kip1. Our data do not support a model in which regulation of p27kip1 function is an important mechanism by which amplified MYCN deregulates cell proliferation in neuroblastoma.

Mutant p53 promotes tumor progression and metastasis by the endoplasmic reticulum UDPase ENTPD5

Significance p53 mutations are the most frequent genetic alteration in cancer and are often indicative of poor patient survival prognosis. The most prevalent missense mutations lead to a “gain of function” (GOF) that actively drives tumor progression, metastasis, and therapy resistance. Our study links the mutant p53 (mutp53) GOF to enhanced N-glycoprotein folding via ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) in the calnexin/calreticulin cycle of the endoplasmic reticulum. Mutp53 thus increases expression of prometastatic cell surface proteins, such as receptors and integrins, not only quantitatively but also qualitatively, with respect to N-glycosylation state. Our study reveals N-glycoprotein quality control in the endoplasmic reticulum as an indispensable mechanism underlying the progression of tumors with GOF mutp53 that could provide new possibilities for treating prognostically challenging p53-mutated cancers. Mutations in the p53 tumor suppressor gene are the most frequent genetic alteration in cancer and are often associated with progression from benign to invasive stages with metastatic potential. Mutations inactivate tumor suppression by p53, and some endow the protein with novel gain of function (GOF) properties that actively promote tumor progression and metastasis. By comparative gene expression profiling of p53-mutated and p53-depleted cancer cells, we identified ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) as a mutant p53 target gene, which functions as a uridine 5′-diphosphatase (UDPase) in the endoplasmic reticulum (ER) to promote the folding of N-glycosylated membrane proteins. A comprehensive pan-cancer analysis revealed a highly significant correlation between p53 GOF mutations and ENTPD5 expression. Mechanistically, mutp53 is recruited by Sp1 to the ENTPD5 core promoter to induce its expression. We show ENTPD5 to be a mediator of mutant p53 GOF activity in clonogenic growth, architectural tissue remodeling, migration, invasion, and lung colonization in an experimental metastasis mouse model. Our study reveals folding of N-glycosylated membrane proteins in the ER as a mechanism underlying the metastatic progression of tumors with mutp53 that could provide new possibilities for cancer treatment.