Xiao-yong Zhang

The Putative Cancer Stem Cell Marker USP22 Is a Subunit of the Human SAGA Complex Required for Activated Transcription and Cell-Cycle Progression

Polycomb genes encode critical regulators of both normal stem cells and cancer stem cells. A gene signature that includes Polycomb genes and additional genes coregulated with Polycomb genes was recently identified. The expression of this signature has been reported to identify tumors with the cancer stem cell phenotypes of aggressive growth, metastasis, and therapy resistance. Most members of this 11 gene signature encode proteins with well-defined roles in human cancer. However, the function of the signature member USP22 remains unknown. We report that USP22 is a previously uncharacterized subunit of the human SAGA transcriptional cofactor complex. Within SAGA, USP22 deubiquitylates histone H2B. Furthermore, USP22 is recruited to specific genes by activators such as the Myc oncoprotein, where it is required for transcription. In support of a functional role within the Polycomb/cancer stem cell signature, USP22 is required for appropriate progression through the cell cycle.

USP22, an hSAGA subunit and potential cancer stem cell marker, reverses the polycomb-catalyzed ubiquitylation of histone H2A

Initial studies of the mammalian hSAGA transcriptional coactivator complex identified the acetyltransferase hGCN5/PCAF as the only known enzymatic subunit. Recently we demonstrated that the ubiquitin hydrolase USP22 comprises a second enzymatic subunit of hSAGA, and that is required for activator-driven transcription. USP22 is expressed with polycomb ubiquitin ligases in an 11 gene signature that defines therapy-resistant tumors. At the biochemical level, these Polycomb proteins function as global transcriptional repressors by catalyzing the ubiquitylation of histone H2A. In yeast, the USP22 homolog functions as a transcriptional coactivator by removing ubiquitin from a distinct core histones, H2B. Given that USP22 is expressed in cancer as part of an 11 gene signature that includes transcriptional repressors which ubiquitylate H2A, it seemed possible that USP22 might activate transcription in part via the deubiquitylation of this same substrate. As reported here, biochemical analysis of the substrate specificity of USP22 reveals that it deubiquitylates histone H2A in addition to H2B. This finding supports a model in which the H2A ubiquitin hydrolase USP22 is coordinately expressed with Polycomb H2A ubiquitin ligases in order that the transcription of certain critical transforming genes be maintained in the face of the global repression mediated by Polycomb.

Regulation of Epstein-Barr Virus Latency Type by the Chromatin Boundary Factor CTCF

ABSTRACT Epstein Barr virus (EBV) can establish distinct latency types with different growth-transforming properties. Type I latency and type III latency can be distinguished by the expression of EBNA2, which has been shown to be regulated, in part, by the EBNA1-dependent enhancer activity of the origin of replication (OriP). Here, we report that CTCF, a chromatin boundary factor with well-established enhancer-blocking activity, binds to EBV sequences between the OriP and the RBP-Jκ response elements of the C promoter (Cp) and regulates transcription levels of EBNA2 mRNA. Using DNA affinity, electrophoretic mobility shift assay, DNase I footprinting, and chromatin immunoprecipitation (ChIP), we found that CTCF binds both in vitro and in vivo to the EBV genome between OriP and Cp, with an ∼50-bp footprint at EBV coordinates 10515 to 10560. Deletion of this CTCF binding site in a recombinant EBV bacterial artificial chromosome (BAC) increased EBNA2 transcription by 3.5-fold compared to a wild-type EBV BAC. DNA affinity and ChIP showed more CTCF binding at this site in type I latency cell lines (MutuI and KemI) than in type III latency cell lines (LCL3456 and Raji). CTCF protein and mRNA expression levels were higher in type I than type III cell lines. Short interfering RNA depletion of CTCF in type I MutuI cells stimulated EBNA2 mRNA levels, while overexpression of CTCF in type III Raji cells inhibited EBNA2 mRNA levels. These results indicate that increased CTCF can repress EBNA2 transcription. We also show that c-MYC, as well as EBNA2, can stimulate CTCF mRNA levels, suggesting that CTCF levels may contribute to B-cell differentiation as well as EBV latency type determination.

Inhibition of the Single Downstream Target BAG1 Activates the Latent Apoptotic Potential of MYC

ABSTRACT Aberrant MYC expression is a common oncogenic event in human cancer. Paradoxically, MYC can either drive cell cycle progression or induce apoptosis. The latent ability of MYC to induce apoptosis has been termed “intrinsic tumor suppressor activity,” and reactivating this apoptotic function in tumors is widely considered a valuable therapeutic goal. As a transcription factor, MYC controls the expression of many downstream targets, and for the majority of these, it remains unclear whether or not they play direct roles in MYC function. To identify the subset of genes specifically required for biological activity, we conducted a screen for functionally important MYC targets and identified BAG1, which encodes a prosurvival chaperone protein. Expression of BAG1 is regulated by MYC in both a mouse model of breast cancer and transformed human cells. Remarkably, BAG1 induction is essential for protecting cells from MYC-induced apoptosis. Ultimately, the synthetic lethality we have identified between MYC overexpression and BAG1 inhibition establishes a new pathway that might be exploited to reactivate the latent apoptotic potential of MYC as a cancer therapy.

Identification of novel targets of MYC whose transcription requires the essential MbII domain.

The MYC oncoprotein is among the most potent regulators of cell cycle progressionand malignant transformation in human cells. Current models suggest that much ofMYC’s role in these processes is related to its ability to regulate the transcription ofdownstream target genes that encode the ultimate effector proteins. In addition to itscarboxy-terminal DNA binding and dimerization domains, an enigmatic motif in theamino terminus termed MbII is required for all of MYC’s biological activities. In spite ofhistorical observations demonstrating the absolute requirement for MbII in thesebiological functions, clues implicating this domain in target gene transcription have onlyrecently appeared. Based on this emerging link between MbII and transcriptionalactivation, we hypothesized that the identification of individual MYC targets whosetransactivation requires MbII would help define the essential downstream effectors ofMYC in transformation and cell cycle progression. In hopes of directly identifying newMbII-dependent MYC target genes, an expression profiling screen was conducted. Thisscreen resulted in our identification of 10 novel downstream targets of MYC. As a proofof principle, we recently demonstrated using RNAi-mediated depletion that one of thesetargets, the metastasis regulator MTA1, is absolutely required for MYC mediatedtransformation. Here we report the identity of these previously uncharacterized MYCtargets and discuss their potential roles in MYC function. In addition, we attempt toreconcile the historical and contemporary evidence linking MbII to transcriptionalactivation.

Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer

Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitts Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors.

Enzymatic assays for assessing histone deubiquitylation activity

While the post-translational modification of histones by the addition of ubiquitin was discovered decades ago, it has only recently been appreciated that the dynamic regulation of histone ubiquitylation patterns is an important mechanism for controlling a variety of biological processes. The processes include transcription, the recognition and repair of genomic damage and DNA replication, among others. Enzymes that catalyze the addition of ubiquitin to histones, such as the polycomb family, have been well-studied. In contrast, the enzymes that remove ubiquitin from histones are less well understood. The assay strategies described here provide a platform for the thorough in vitro and in vivo analysis of histone deubiquitylation. In some cases, these poorly characterized enzymes are likely to provide new opportunities for therapeutic targeting and a detailed understanding of their biochemical and biological activities is a prerequisite to these clinical advances.

Abstract 1256: Multi-focal control of mitochondrial gene expression by oncogenic MYC provides effective therapeutic targets in cancer

The MYC transcription factor is the most dramatically overexpressed gene product in human cancer. However, defining the MYC-driven transcriptome critical to malignant transformation has remained a challenge, in part because MYC controls several thousand genes. Consequently, little progress has been made in the identification of sensitive nodes downstream of MYC that might be targeted therapeutically in order to interrupt its oncogenic signaling. To overcome this impediment, we performed a screen for downstream MYC effector pathways that are selectively linked to MYC9s functional properties. Via this strategy, we identified the mitochondrial RNA polymerase (POLRMT) as a direct downstream target of MYC. POLRMT induction by MYC regulates transcription of the mitochondrial genome, as well as mitochondrial DNA replication. Recent studies have shown that POLRMT also interacts with a mitochondrial ribosomal RNA methyltransferase TFB1M to regulate mitochondrial ribosome assembly. Notably, blocking induction of POLRMT (or TFB1M) by MYC converts the cellular response to MYC activation from enhanced cell cycle progression to apoptotic cell death. Of immediate clinical relevance, compounds blocking mitochondrial gene expression at any of several steps cause acute synthetic lethality with oncogenic levels of MYC. Among these compounds, the mitochondrial targeting antibiotic Tigecycline is FDA-approved and well-tolerated in patients. Tigecycline eliminates tumor formation and dramatically improves survival in our animal model of MYC-driven lymphoma. Evidence presented here demonstrates that uncoupling of MYC9s nuclear and mitochondrial transcriptomes exposes a point of exquisite vulnerability in tumor cells. Although targeting MYC as a therapeutic strategy has proven to be challenging in the past, our identification of a new node of sensitivity in MYC-driven cancers offers a potential for broad therapeutic implications. Citation Format: Amanda Taylor, Clare Adams, Xiao-yong Zhang, Victoria Gennaro, Justin Cotney, Gerald Shadel, Craig Cameron, Christine Eischen, Steven McMahon. Multi-focal control of mitochondrial gene expression by oncogenic MYC provides effective therapeutic targets in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1256.

Abstract 4553: Identification of CD30/TNFRSF8 as a primary target of the Myc oncoprotein and potential biomarker of Myc-driven cancer

Pathological activation of the transcription factor Myc induces a multitude of human malignancies. In Myc-driven tumor progression, there is a robust dependence on Myc activity; however, strategies developed to explicitly modulate Myc function in cancer have yielded little clinical success. Patients harboring Myc-dependent tumors would benefit from a therapy that circumvents molecular targeting of Myc expression and instead directs against a distinct downstream effector. Our preliminary data from an unbiased expression-profiling screen identify CD30 as a primary target of the Myc oncoprotein. CD30 is a member of the tumor necrosis factor superfamily and resides on the extracellular membrane; it is highly expressed in several lymphomas while acutely restricted in normal cells. Within the last few decades, CD30 has emerged as a diagnostic marker and therapeutic target of many lymphoproliferative diseases. However, the underlying mechanisms that regulate CD30 expression remain unclear. We report that CD30 is regulated at the transcriptional level by Myc. Through a genetic and biochemical approach, we define CD30 induction in a MYC-dependent manner across a variety of human cell lines of both lymphomagenic and non-lymphomagenic origin. Furthermore, deliberate modulation of Myc-activity demonstrates a positive correlation with CD30 surface protein expression in multiple malignancy models. With CD30 induction as a potential mechanism by which to identify and target Myc-driven oncogenesis, we can reshape detection and treatment of Myc-regulated cancer. Citation Format: Victoria Gennaro, Xiao-yong Zhang, Lauren DeSalle, Duonan Yu, Andrei Thomas-Tikhonenko, Christopher Vakoc, Steven B. McMahon. Identification of CD30/TNFRSF8 as a primary target of the Myc oncoprotein and potential biomarker of Myc-driven cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4553.

Abstract A40: CD30: A therapeutic target of MYC-driven cancer

The MYC oncogene induces a multitude of human malignancies and has been well characterized as a critical driver of select lymphoma subtypes. In MYC-amplified tumor progression, there is a robust dependence on MYC activity; however, strategies developed to modulate MYC function in cancer have yielded little success. Patients harboring these MYC-driven tumors would benefit from a directed therapy that circumvents explicit molecular targeting of MYC expression. Our preliminary data from an unbiased expression-profiling screen identified CD30 as a primary downstream target of the MYC oncoprotein. CD30 is a member of the tumor necrosis factor superfamily and resides on the extracellular membrane; it is highly expressed in many lymphomas while acutely restricted in normal cells. Within the last few decades, CD30 has emerged as a diagnostic marker and therapeutic target of many lymphoproliferative disorders and diseases. The biological function of CD30 is pleiotropic: CD30 stimulation is reportedly linked to cell cycle arrest, apoptosis, and proliferation through activation of the pro-survival transcription factor, NF-kB. However, the underlying mechanisms that regulate CD30 expression remain unclear. CD30-driven oncogenic pathways phenocopy those initiated by MYC, suggesting CD309s potential status as an effector of MYC-driven tumorigenic events. Here we report that CD30 is regulated at the transcriptional level by MYC. Through a genetic and biochemical approach, we define CD30 induction in a MYC-dependent manner across a variety of human cell lines of both lymphomagenic and non-lymphomagenic origin. Furthermore, in a murine lymphoma model where tumor formation is driven by a conditional allele of MYC, CD30 transcript levels are tightly correlated with MYC activity. Ultimately, CD30 induction offers a novel therapeutic avenue by which to target MYC-driven oncogenesis and has the potential to reshape detection and treatment of MYC-regulated cancer. Citation Format: Victoria Gennaro, Xiao-yong Zhang, Lauren DeSalle, Duonan Yu, Andrei Thomas-Tikhonenko, Steven McMahon. CD30: A therapeutic target of MYC-driven cancer. [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 A40.