Showing papers by "Michael R. Green published in 2012"

Constitutive AP-1 Activity and EBV Infection Induce PD-L1 in Hodgkin Lymphomas and Posttransplant Lymphoproliferative Disorders: Implications for Targeted Therapy

Abstract: Purpose: Programmed cell death ligand 1 (PD-L1) is a molecule expressed on antigen-presenting cells that engages the PD-1 receptor on T cells and inhibits T-cell receptor signaling. The PD-1 axis can be exploited by tumor cells to dampen host antitumor immune responses and foster tumor cell survival. PD-1 blockade has shown promise in multiple malignancies but should be directed toward patients in whom it will be most effective. In recent studies, we found that the chromosome 9p24.1 amplification increased the gene dosage of PD-L1 and its induction by JAK2 in a subset of patients with classical Hodgkin lymphoma (cHL). However, cHLs with normal 9p24.1 copy numbers also expressed detectable PD-L1, prompting analyses of additional PD-L1 regulatory mechanisms. Experimental Design: Herein, we utilized immunohistochemical, genomic, and functional analyses to define alternative mechanisms of PD-L1 activation in cHL and additional EBV+ lymphoproliferative disorders. Results: We identified an AP-1–responsive enhancer in the PD-L1 gene. In cHL Reed–Sternberg cells, which exhibit constitutive AP-1 activation, the PD-L1 enhancer binds AP-1 components and increases PD-L1 promoter activity. In addition, we defined Epstein–Barr virus (EBV) infection as an alternative mechanism for PD-L1 induction in cHLs with diploid 9p24.1. PD-L1 was also expressed by EBV-transformed lymphoblastoid cell lines as a result of latent membrane protein 1–mediated, JAK/STAT-dependent promoter and AP-1–associated enhancer activity. In addition, more than 70% of EBV+ posttransplant lymphoproliferative disorders expressed detectable PD-L1. Conclusions: AP-1 signaling and EBV infection represent alternative mechanisms of PD-L1 induction and extend the spectrum of tumors in which to consider PD-1 blockade. Clin Cancer Res; 18(6); 1611–8. ©2012 AACR.

Characterization of Enhancer Function from Genome-Wide Analyses

Abstract: There has been a recent surge in the use of genome-wide methodologies to identify and annotate the transcriptional regulatory elements in the human genome. Here we review some of these methodologies and the conceptual insights about transcription regulation that have been gained from the use of genome-wide studies. It has become clear that the binding of transcription factors is itself a highly regulated process, and binding does not always appear to have functional consequences. Numerous properties have now been associated with regulatory elements that may be useful in their identification. Several aspects of enhancer function have been shown to be more widespread than was previously appreciated, including the highly combinatorial nature of transcription factor binding, the postinitiation regulation of many target genes, and the binding of enhancers at early stages to maintain their competence during development. Going forward, the integration of multiple genome-wide data sets should become a standard approach to elucidate higher-order regulatory interactions.

The Blk pathway functions as a tumor suppressor in chronic myeloid leukemia stem cells

Abstract: A therapeutic strategy for treating cancer is to target and eradicate cancer stem cells (CSCs) without harming their normal stem cell counterparts. The success of this approach relies on the identification of molecular pathways that selectively regulate CSC function. Using BCR-ABL-induced chronic myeloid leukemia (CML) as a disease model for CSCs, we show that BCR-ABL downregulates the Blk gene (encoding B-lymphoid kinase) through c-Myc in leukemic stem cells (LSCs) in CML mice and that Blk functions as a tumor suppressor in LSCs but does not affect normal hematopoietic stem cells (HSCs) or hematopoiesis. Blk suppresses LSC function through a pathway involving an upstream regulator, Pax5, and a downstream effector, p27. Inhibition of this Blk pathway accelerates CML development, whereas increased activity of the Blk pathway delays CML development. Blk also suppresses the proliferation of human CML stem cells. Our results show the feasibility of selectively targeting LSCs, an approach that should be applicable to other cancers.

Transcription factor ATF5 is required for terminal differentiation and survival of olfactory sensory neurons

Abstract: Activating transcription factor 5 (ATF5) is a member of the ATF/cAMP response element-binding family of transcription factors, which compose a large group of basic region leucine zipper proteins whose members mediate diverse transcriptional regulatory functions. ATF5 has a well-established prosurvival activity and has been found to be overexpressed in several human cancers, in particular glioblastoma. However, the role(s) of ATF5 in development and normal physiology are unknown. Here we address this issue by deriving and characterizing homozygous Atf5 knockout mice. We find that Atf5−/− pups die neonatally, which, as explained below, is consistent with an olfactory defect resulting in a competitive suckling deficit. We show that Atf5 is highly expressed in olfactory sensory neurons (OSNs) in the main olfactory epithelium starting from embryonic stage 11.5 through adulthood. Immunostaining experiments with OSN-specific markers reveal that ATF5 is expressed in some immature OSNs and in all mature OSNs. Expression profiling and immunostaining experiments indicate that loss of Atf5 leads to a massive reduction in mature OSNs resulting from a differentiation defect and the induction of apoptosis. Ectopic expression of Atf5 in neural progenitor cells induces expression of multiple OSN-specific genes. Collectively, our results suggest a model in which Atf5 is first expressed in immature OSNs and the resultant ATF5 functions to promote differentiation into mature OSNs. Thus, ATF5 is required for terminal differentiation and survival of OSNs.

Non-canonical TAF complexes regulate active promoters in human embryonic stem cells

Abstract: The general transcription factor TFIID comprises the TATA-box-binding protein (TBP) and approximately 14 TBP-associated factors (TAFs). Here we find, unexpectedly, that undifferentiated human embryonic stem cells (hESCs) contain only six TAFs (TAFs 2, 3, 5, 6, 7 and 11), whereas following differentiation all TAFs are expressed. Directed and global chromatin immunoprecipitation analyses reveal an unprecedented promoter occupancy pattern: most active genes are bound by only TAFs 3 and 5 along with TBP, whereas the remaining active genes are bound by TBP and all six hESC TAFs. Consistent with these results, hESCs contain a previously undescribed complex comprising TAFs 2, 6, 7, 11 and TBP. Altering the composition of hESC TAFs, either by depleting TAFs that are present or ectopically expressing TAFs that are absent, results in misregulated expression of pluripotency genes and induction of differentiation. Thus, the selective expression and use of TAFs underlies the ability of hESCs to self-renew. DOI: http://dx.doi.org/10.7554/eLife.00068.001

A synthetic interaction screen identifies factors selectively required for proliferation and TERT transcription in p53-deficient human cancer cells.

Abstract: Numerous genetic and epigenetic alterations render cancer cells selectively dependent on specific genes and regulatory pathways, and represent potential vulnerabilities that can be therapeutically exploited. Here we describe an RNA interference (RNAi)–based synthetic interaction screen to identify genes preferentially required for proliferation of p53-deficient (p53−) human cancer cells. We find that compared to p53-competent (p53+) human cancer cell lines, diverse p53− human cancer cell lines are preferentially sensitive to loss of the transcription factor ETV1 and the DNA damage kinase ATR. In p53− cells, RNAi–mediated knockdown of ETV1 or ATR results in decreased expression of the telomerase catalytic subunit TERT leading to growth arrest, which can be reversed by ectopic TERT expression. Chromatin immunoprecipitation analysis reveals that ETV1 binds to a region downstream of the TERT transcriptional start-site in p53− but not p53+ cells. We find that the role of ATR is to phosphorylate and thereby stabilize ETV1. Our collective results identify a regulatory pathway involving ETV1, ATR, and TERT that is preferentially important for proliferation of diverse p53− cancer cells.

Analysis of Gal4-directed transcription activation using Tra1 mutants selectively defective for interaction with Gal4

Abstract: Promoter-specific transcriptional activators (activators) stimulate transcription through direct interactions with one or more components of the transcription machinery, termed the "target." The identification of direct in vivo targets of activators has been a major challenge. Previous studies have provided evidence that the Tra1 subunit of the yeast SAGA (Spt-Ada-Gcn5-acetyltransferase) complex is the target of the yeast activator Gal4. However, several other general transcription factors, in particular the mediator complex, have also been implicated as Gal4 targets. Here we perform a large-scale genetic screen to derive and characterize tra1 alleles that are selectively defective for interaction with Gal4 in vivo [Gal4 interaction defective (GID) mutants]. In contrast to WT Tra1, Tra1 GID mutants are not recruited by Gal4 to the promoter and cannot support Gal4-directed transcription, demonstrating the essentiality of the Gal4-Tra1 interaction. In yeast strains expressing a Tra1 GID mutant, binding of Gal4 to the promoter is unexpectedly also diminished, indicating that Gal4 and Tra1 bind cooperatively. Consistent with cooperative binding, we demonstrate that the Gal4-Tra1 interaction occurs predominantly on the promoter and not off DNA. Finally, we show that although Tra1 is targeted by other activators, these interactions are unaffected by GID mutations, revealing an unanticipated specificity of the Gal4-Tra1 interaction.

Srpx for treatment of cancer

Abstract: Methods of treating a tumor in a subject include identifying a subject having, at risk for, or suspected of having a tumor, and administering to the subject an effective amount of an SRPX.