Genome maintenance in retinoblastoma: Implications for therapeutic vulnerabilities.

Retinoblastoma (RB) is a pediatric ocular malignancy that is initiated mostly by biallelic inactivation of the RB transcriptional corepressor 1 (RB1) tumor suppressor gene in the developing retina. Unlike the prevailing prediction based on multiple studies involving RB1 gene disruption in experimental models, human RB tumors have been demonstrated to possess a relatively stable genome, characterized by a low mutation rate and a few recurrent chromosomal alterations related to somatic copy number changes. This suggests that RB may harbor heightened genome maintenance mechanisms to counteract or compensate for the risk of massive genome instability, which can potentially be driven by the early RB1 loss as a tumor-initiating event. Although the genome maintenance mechanisms might have been evolved to promote RB cell survival by preventing lethal genomic defects, emerging evidence suggests that the dependency of RB cells on these mechanisms also exposes their unique vulnerability to chemotherapy, particularly when the genome maintenance machineries are tumor cell-specific. This review summarizes the genome maintenance mechanisms identified in RB, including findings on the roles of chromatin regulators in DNA damage response/repair and protein factors involved in maintaining chromosome stability and promoting survival in RB. In addition, advantages and challenges for exploiting these therapeutic vulnerabilities in RB are discussed.

Targeting of histone methyltransferase DOT1L plays a dual role in chemosensitization of retinoblastoma cells and enhances the efficacy of chemotherapy.

Aberrant and exclusive expression of chromatin regulators in retinoblastoma (RB) in contrast to terminally differentiated normal retina presents a unique opportunity of selective targeting for RB. However, precise roles of these chromatin regulators in RB development and their potential as therapeutic targets have not been defined thoroughly. Here, we report that targeting of disruptor of telomeric silencing 1-like (DOT1L), a histone H3K79 methyltransferase, sensitizes RB cells to chemotherapeutic drugs by impairing the DNA damage response and thereby potentiating apoptosis while it is largely inefficacious as a single-agent therapy. Moreover, we identified high mobility group AT-hook 2 (HMGA2) as a novel DOT1L target gene in RB cells and found that its aberrant expression is dependent on DOT1L. As HMGA2 depletion reduced CHK1 phosphorylation during DNA damage response and augmented the drug sensitivity in RB cells, our results suggested that DOT1L targeting has a dual role in chemosensitization of RB cells by directly interfering with the immediate involvement of DOT1L in early DNA damage response upon genotoxic insults and also by downregulating the expression of HMGA2 as a rather late effect of DOT1L inhibition. Furthermore, we provide the first preclinical evidence demonstrating that combined therapy with a DOT1L inhibitor significantly improves the therapeutic efficacy of etoposide in murine orthotopic xenografts of RB by rendering the response to etoposide more potent and stable. Taken together, these results support the therapeutic benefits of DOT1L targeting in combination with other chemotherapeutic agents in RB, with mechanistic insights into how DOT1L targeting can improve the current chemotherapy in an RB cell-selective manner.

Chromatin regulators in retinoblastoma: Biological roles and therapeutic applications.

Retinoblastoma (RB) is a pediatric ocular tumor mostly occurring due to the biallelic loss of RB1 gene in the developing retina. Early studies of genomic aberrations in RB have provided a valuable insight into how RB can progress following the tumor-initiating RB1 mutations and have established a notion that inactivation of RB1 gene is critical to initiate RB but this causative genetic lesion alone is not sufficient for malignant progression. With the advent of high-throughput sequencing technologies, we now have access to the comprehensive genomic and epigenetic landscape of RB and have come to appreciate that RB tumorigenesis requires both genetic and epigenetic alterations that might be directly or indirectly driven by RB1 loss. This integrative perspective on RB tumorigenesis has inspired research efforts to better understand the types and functions of epigenetic mechanisms contributing to RB development, leading to the identification of multiple epigenetic regulators misregulated in RB in recent years. A complete understanding of the intricate network of genetic and epigenetic factors in modulation of gene expression during RB tumorigenesis remains a major challenge but would be crucial to translate these findings into therapeutic interventions. In this review, we will provide an overview of chromatin regulators identified to be misregulated in human RB among the numerous epigenetic factors implicated in RB development. For a subset of these chromatin regulators, recent findings on their functions in RB development and potential therapeutic applications are discussed.

UHRF1 downmodulation enhances antitumor effects of histone deacetylase inhibitors in retinoblastoma by augmenting oxidative stress-mediated apoptosis.

Identification of new genetic pathways or molecular targets that sensitize cancer cells to chemotherapeutic drugs may improve the efficacy of current chemotherapy. Here, we report that downmodulation of UHRF1 (ubiquitin-like with PHD and RING finger domains 1) in retinoblastoma (RB) cells increases the sensitivity to histone deacetylase (HDAC) inhibitors, augmenting apoptotic cell death. We found that UHRF1 depletion downregulates two redox-responsive genes GSTA4 (glutathione S-transferase α4) and TXN2 (thioredoxin-2) in RB cells, and increases the basal level of intracellular oxidative stress. Antioxidant treatment significantly reduced both basal and HDAC inhibitor-induced DNA damage and apoptosis in UHRF1-depleted cells. Knockdown of GSTA4 or TXN2 sensitized RB cells to HDAC inhibitors, demonstrating that GSTA4 and TXN2 play key roles in redox homeostasis in RB cells and the susceptibility to HDAC inhibitor treatment upon UHRF1 depletion. In human primary RB, GSTA4 and TXN2 proteins were found to be mostly elevated along with high UHRF1 expression. In addition to augmentation of apoptosis in UHRF1-depleted RB cells, we also show that UHRF1 downmodulation derepresses the expression of photoreceptor-specific genes in RB cells in cooperation with a HDAC inhibitor MS-275 and promotes neuron-like differentiation. However, further investigation revealed that the enhanced growth-inhibitory effects of MS-275 in UHRF1-depleted cells were still mainly due to robust apoptosis induction rather than differentiation-mediated growth arrest. Consistent with our findings, UHRF1 depletion in RB cells increased the therapeutic efficacy of MS-275 in murine orthotopic xenografts. These results provide a novel basis for potential benefits of UHRF1 targeting for RB treatment.

UHRF1 depletion sensitizes retinoblastoma cells to chemotherapeutic drugs via downregulation of XRCC4.

UHRF1 (ubiquitin-like with PHD and ring finger domains 1) is highly expressed in various human cancers including retinoblastoma, and associated with tumor-promoting effects such as inhibition of apoptosis and high proliferation. However, the molecular mechanisms underlying tumor-promoting functions of UHRF1 in retinoblastoma still remain elusive. Here, we show that stable knockdown of UHRF1 renders retinoblastoma cells sensitized to conventional chemotherapeutic drugs such as etoposide and camptothecin, resulting in enhanced DNA damage and apoptotic cell death. We found that UHRF1-depleted retinoblastoma cells can recognize DNA damages normally but have markedly low expression of XRCC4 (X-ray repair cross complementing 4) among the components of nonhomologous end-joining (NHEJ) repair complex. Conversely, overexpression of UHRF1 increased the XRCC4 expression and stable knockdown of XRCC4 sensitized retinoblastoma cells to etoposide treatment, suggesting that XRCC4 is a key mediator for the drug sensitivity upon UHRF1 depletion in retinoblastoma cells. Consistent with the findings, chromatin association of DNA ligase IV in response to acute DNA damage was found to be significantly reduced in UHRF1-depleted retinoblastoma cells and functional complementation for XRCC4 in UHRF1-depleted cells attenuated the drug sensitivity, demonstrating that XRCC4 downregulation in UHRF1-depleted cells impaired DNA repair and consequently induced robust apoptosis upon genotoxic drug treatment. In human primary retinoblastoma, high expression of UHRF1 and XRCC4 could be detected, and elevated XRCC4 expression correlated with reduced apoptosis markers, implying that UHRF1-mediated XRCC4 upregulation under pathophysiological conditions triggered by RB1 gene inactivation may confer protection against endogenous DNA damages that arise during retinoblastoma development. Taken together, these results present a new mechanistic insight into how UHRF1 mediates its tumor-promoting functions in retinoblastoma, and also provide a basis for UHRF1 targeting to improve the efficacy of current chemotherapy for retinoblastoma treatment.

Functional dissection of the role of UHRF1 in the regulation of retinoblastoma methylome.

UHRF1 (ubiquitin-like with PHD and RING finger domains 1) is a critical regulator for DNA methylation, and its frequent overexpression in human cancers has been associated with tumor-promoting effects. However, whether the overexpressed UHRF1 contributes to the establishment and maintenance of tumor methylomes and whether this process can affect the tumorigenesis remain unclear. In this study, we show that UHRF1 is highly expressed in retinoblastoma, and genomes of human primary retinoblastoma and cell lines have differential DNA methylation patterns compared with those of normal retina, characterized by lower global methylation and higher promoter methylation of tumor suppressors. However, our genome-wide DNA methylation study uncovers that UHRF1 down-modulation in retinoblastoma cells exerts minor effects on the existing methylation patterns at both bulk genome and individual gene loci, suggesting that retinoblastoma methylome is primarily maintained by other mechanisms. Furthermore, using two murine retinoblastoma models, we found that high UHRF1 expression does not alter global methylation levels in both premalignant neonatal retina and retinoblastoma tumors, implying that DNA hypomethylation may not be an early mechanism driving retinoblastoma tumorigenesis unlike what has been proposed for other types of cancer. These results suggest that tumor-promoting functions of UHRF1 in retinoblastoma are largely independent of its role in DNA methylation.

Coupling of T cell receptor specificity to natural killer T cell development by bivalent histone H3 methylation.

The fidelity of T cell immunity depends greatly on coupling T cell receptor signaling with specific T cell effector functions. Here, we describe a chromatin-based mechanism that enables integration of TCR specificity into definite T cell lineage commitment. Using natural killer T cells (iNKT cell) as a model of a T cell subset that differentiates in response to specific TCR signaling, we identified a key role of histone H3 lysine 27 trimethylation (H3K27me3) in coupling iNKT cell TCR specificity with the generation of iNKT cells. We found that the Zbtb16/PLZF gene promoter that drives iNKT cell differentiation possesses a bivalent chromatin state characterized by the simultaneous presence of negative and positive H3K27me3 and H3K4me3 modifications. Depletion of H3K27me3 at the Zbtb16/PLZF promoter leads to uncoupling of iNKT cell development from TCR specificity and is associated with accumulation of iNKT-like CD4(+) cells that express a non-iNKT cell specific T cell repertoire. In turn, stabilization of H3K27me3 leads to a drastic reduction of the iNKT cell population. Our data suggest that H3K27me3 levels at the bivalent Zbtb16/PLZF gene define a threshold enabling precise coupling of TCR specificity to lineage commitment.

Nuclear cyclin D1/CDK4 kinase regulates CUL4 expression and triggers neoplastic growth via activation of the PRMT5 methyltransferase.

Cyclin D1 elicits transcriptional effects through inactivation of the retinoblastoma protein and direct association with transcriptional regulators. The current work reveals a molecular relationship between cyclin D1/CDK4 kinase and protein arginine methyltransferase 5 (PRMT5), an enzyme associated with histone methylation and transcriptional repression. Primary tumors of a mouse lymphoma model exhibit increased PRMT5 methyltransferase activity and histone arginine methylation. Analyses demonstrate that MEP50, a PRMT5 coregulatory factor, is a CDK4 substrate, and phosphorylation increases PRMT5/MEP50 activity. Increased PRMT5 activity mediates key events associated with cyclin D1-dependent neoplastic growth, including CUL4 repression, CDT1 overexpression, and DNA rereplication. Importantly, human cancers harboring mutations in Fbx4, the cyclin D1 E3 ligase, exhibit nuclear cyclin D1 accumulation and increased PRMT5 activity.

Nuclear cyclin D1: an oncogenic driver in human cancer.

Perturbations in the regulation of the core cell cycle machinery are frequently observed in human cancers. Cyclin D1 which functions as a mitogenic sensor and allosteric activator of CDK4/6, is one of the more frequently altered cell cycle regulators in cancers. Cyclin D1 is frequently overexpressed in cancers and its overexpression can be attributed to many factors including increased transcription, translation, and protein stability. Although cyclin D1 overexpression is clearly implicated in the affected cancers, overexpression of cyclin D1 is not sufficient to drive oncogenic transformation. Rather, emerging evidence suggests that nuclear retention of cyclin D1 resulting from altered nuclear trafficking and proteolysis is critical for the manifestation of its oncogenicity. This review provides a brief overview of current data documenting various mechanisms underlying aberrant cyclin D1 regulation in human cancers and their impact on neoplastic transformation.

BWMK1, a rice mitogen-activated protein kinase, locates in the nucleus and mediates pathogenesis-related gene expression by activation of a transcription factor.

Mitogen-activated protein kinase (MAPK) cascades are known to transduce plant defense signals, but the downstream components of the MAPK have as yet not been elucidated. Here, we report an MAPK from rice (Oryza sativa), BWMK1, and a transcription factor, OsEREBP1, phosphorylated by the kinase. The MAPK carries a TDY phosphorylation motif instead of the more common TEY motif in its kinase domain and has an unusually extended C-terminal domain that is essential to its kinase activity and translocation to the nucleus. The MAPK phosphorylates OsEREBP1 that binds to the GCC box element (AGCCGCC) of the several basic pathogenesis-related gene promoters, which in turn enhances DNA-binding activity of the factor to the cis element in vitro. Transient co-expression of the BWMK1 and OsEREBP1 in Arabidopsis protoplasts elevates the expression of the beta-glucuronidase reporter gene driven by the GCC box element. Furthermore, transgenic tobacco (Nicotiana tabacum) plants overexpressing BWMK1 expressed many pathogenesis-related genes at higher levels than wild-type plants with an enhanced resistance to pathogens. These findings suggest that MAPKs contribute to plant defense signal transduction by phosphorylating one or more transcription factors.

Mlo, a modulator of plant defense and cell death, is a novel calmodulin-binding protein. Isolation and characterization of a rice Mlo homologue.

Transient influx of Ca(2+) constitutes an early event in the signaling cascades that trigger plant defense responses. However, the downstream components of defense-associated Ca(2+) signaling are largely unknown. Because Ca(2+) signals are mediated by Ca(2+)-binding proteins, including calmodulin (CaM), identification and characterization of CaM-binding proteins elicited by pathogens should provide insights into the mechanism by which Ca(2+) regulates defense responses. In this study, we isolated a gene encoding rice Mlo (Oryza sativa Mlo; OsMlo) using a protein-protein interaction-based screening of a cDNA expression library constructed from pathogen-elicited rice suspension cells. OsMlo has a molecular mass of 62 kDa and shares 65% sequence identity and scaffold topology with barley Mlo, a heptahelical transmembrane protein known to function as a negative regulator of broad spectrum disease resistance and leaf cell death. By using gel overlay assays, we showed that OsMlo produced in Escherichia coli binds to soybean CaM isoform-1 (SCaM-1) in a Ca(2+)-dependent manner. We located a 20-amino acid CaM-binding domain (CaMBD) in the OsMlo C-terminal cytoplasmic tail that is necessary and sufficient for Ca(2+)-dependent CaM complex formation. Specific binding of the conserved CaMBD to CaM was corroborated by site-directed mutagenesis, a gel mobility shift assay, and a competition assay with a Ca(2+)/CaM-dependent enzyme. Expression of OsMlo was strongly induced by a fungal pathogen and by plant defense signaling molecules. We propose that binding of Ca(2+)-loaded CaM to the C-terminal tail may be a common feature of Mlo proteins.