Insights into LINE-1 reverse transcription guide therapy development.

Subsets of long interspersed nuclear element 1 (LINE-1) retrotransposons can 'retrotranspose' throughout the human genome at a cost to host cell fitness, as observed in some cancers. Pharmacological inhibition of LINE-1 retrotransposition requires a comprehensive understanding of the LINE-1 ORF2p reverse transcriptase. Two recent publications, by Thawani et al. and Baldwin et al., report structures of LINE-1 ORF2p and address long-standing mechanistic gaps regarding LINE-1 retrotransposition. Both studies will be critical to design new specific inhibitors of the LINE-1 ORF2p reverse transcriptase.

Modeling methyl-sensitive transcription factor motifs with an expanded epigenetic alphabet.

BACKGROUND: Transcription factors bind DNA in specific sequence contexts. In addition to distinguishing one nucleobase from another, some transcription factors can distinguish between unmodified and modified bases. Current models of transcription factor binding tend not to take DNA modifications into account, while the recent few that do often have limitations. This makes a comprehensive and accurate profiling of transcription factor affinities difficult. RESULTS: Here, we develop methods to identify transcription factor binding sites in modified DNA. Our models expand the standard A/C/G/T DNA alphabet to include cytosine modifications. We develop Cytomod to create modified genomic sequences and we also enhance the MEME Suite, adding the capacity to handle custom alphabets. We adapt the well-established position weight matrix (PWM) model of transcription factor binding affinity to this expanded DNA alphabet. Using these methods, we identify modification-sensitive transcription factor binding motifs. We confirm established binding preferences, such as the preference of ZFP57 and C/EBPß for methylated motifs and the preference of c-Myc for unmethylated E-box motifs. CONCLUSIONS: Using known binding preferences to tune model parameters, we discover novel modified motifs for a wide array of transcription factors. Finally, we validate our binding preference predictions for OCT4 using cleavage under targets and release using nuclease (CUT&RUN) experiments across conventional, methylation-, and hydroxymethylation-enriched sequences. Our approach readily extends to other DNA modifications. As more genome-wide single-base resolution modification data becomes available, we expect that our method will yield insights into altered transcription factor binding affinities across many different modifications.

Retroelement decay by the exonuclease XRN1 is a viral mimicry dependency in cancer.

Viral mimicry describes the immune response induced by endogenous stimuli such as double-stranded RNA (dsRNA) from endogenous retroelements. Activation of viral mimicry has the potential to kill cancer cells or augment anti-tumor immune responses. Here, we systematically identify mechanisms of viral mimicry adaptation associated with cancer cell dependencies. Among the top hits is the RNA decay protein XRN1 as an essential gene for the survival of a subset of cancer cell lines. XRN1 dependency is mediated by mitochondrial antiviral signaling protein and protein kinase R activation and is associated with higher levels of cytosolic dsRNA, higher levels of a subset of Alus capable of forming dsRNA, and higher interferon-stimulated gene expression, indicating that cells die due to induction of viral mimicry. Furthermore, dsRNA-inducing drugs such as 5-aza-2'-deoxycytidine and palbociclib can generate a synthetic dependency on XRN1 in cells initially resistant to XRN1 knockout. These results indicate that XRN1 is a promising target for future cancer therapeutics.

Endogenous Retroelements and the Viral Mimicry Response in Cancer Therapy and Cellular Homeostasis.

Features of the cancer epigenome distinguish cancers from their respective cell of origin and establish therapeutic vulnerabilities that can be exploited through pharmacologic inhibition of DNA- or histone-modifying enzymes. Epigenetic therapies converge with cancer immunotherapies through "viral mimicry," a cellular state of active antiviral response triggered by endogenous nucleic acids often derived from aberrantly transcribed endogenous retrotransposons. This review describes the initial characterization and expansion of viral mimicry-inducing approaches as well as features that "prime" cancers for viral mimicry induction. Increased understanding of viral mimicry in therapeutic contexts suggests potential physiologic roles in cellular homeostasis. SIGNIFICANCE: Recent literature establishes elevated cytosolic double strand RNA (dsRNA) levels as a cancer-specific therapeutic vulnerability that can be elevated by viral mimicry-inducing therapies beyond tolerable thresholds to induce antiviral signaling and increase dependence on dsRNA stress responses mediated by ADAR1. Improved understanding of viral mimicry signaling and tolerance mechanisms reveals synergistic treatment combinations with epigenetic therapies that include inhibition of BCL2, ADAR1, and immune checkpoint blockade. Further characterization of viral mimicry tolerance may identify contexts that maximize efficacy of conventional cancer therapies.

DNA hypomethylating agents increase activation and cytolytic activity of CD8+ T cells.

We demonstrate that DNA hypomethylating agent (HMA) treatment can directly modulate the anti-tumor response and effector function of CD8+ T cells. In vivo HMA treatment promotes CD8+ T cell tumor infiltration and suppresses tumor growth via CD8+ T cell-dependent activity. Ex vivo, HMAs enhance primary human CD8+ T cell activation markers, effector cytokine production, and anti-tumor cytolytic activity. Epigenomic and transcriptomic profiling shows that HMAs vastly regulate T cell activation-related transcriptional networks, culminating with over-activation of NFATc1 short isoforms. Mechanistically, demethylation of an intragenic CpG island immediately downstream to the 3' UTR of the short isoform was associated with antisense transcription and alternative polyadenylation of NFATc1 short isoforms. High-dimensional single-cell mass cytometry analyses reveal a selective effect of HMAs on a subset of human CD8+ T cell subpopulations, increasing both the number and abundance of a granzyme Bhigh, perforinhigh effector subpopulation. Overall, our findings support the use of HMAs as a therapeutic strategy to boost anti-tumor immune response.

Spliceosome-Targeted Therapies Induce dsRNA Responses.

In a recent issue of Cell, Bowling et al. describe a mechanism by which spliceosome-targeted therapies result in intron-containing transcripts that form double-stranded RNAs (dsRNAs), thereby activating tumor antiviral signaling (viral mimicry) and downstream adaptive immunity.

DNA Methylation Profiling of Premalignant Lesions as a Path to Ovarian Cancer Early Detection.

Genome-wide DNA methylation profiles of serous tubal intraepithelial carcinomas (STIC) resemble methylation profiles of high-grade serous ovarian carcinomas (HGSOC) more closely than normal fallopian tube epithelium. While STICs and HGSOCs share subsets of common hypermethylated regions, DNA methylation can distinguish STICs from HGSOCs to provide proof-of-principle that DNA methylation can identify HGSOC initiation.See related article by Pisanic et al., p. 6310.

An open-label, phase II multicohort study of an oral hypomethylating agent CC-486 and durvalumab in advanced solid tumors.

PURPOSE: To evaluate whether administration of the oral DNA hypomethylating agent CC-486 enhances the poor response rate of immunologically 'cold' solid tumors to immune checkpoint inhibitor durvalumab. EXPERIMENTAL DESIGN: PD-L1/PD-1 inhibitor naïve patients with advanced microsatellite stable colorectal cancer; platinum resistant ovarian cancer; and estrogen receptor positive, HER2 negative breast cancer were enrolled in this single-institution, investigator-initiated trial. Two 28 day regimens, regimen A (CC-486 300 mg QD Days 1-14 (cycles 1-3 only) in combination with durvalumab 1500 mg intravenous day 15) and regimen B (CC-486 100 mg QD days 1-21 (cycle 1 and beyond), vitamin C 500 mg once a day continuously and durvalumab 1500 mg intravenous day 15) were investigated. Patients underwent paired tumor biopsies and serial peripheral blood mononuclear cells (PBMCs) collection for immune-profiling, transcriptomic and epigenomic analyzes. RESULTS: A total of 28 patients were enrolled, 19 patients treated on regimen A and 9 on regimen B. The combination of CC-486 and durvalumab was tolerable. Regimen B, with a lower dose of CC-486 extended over a longer treatment course, showed less grade 3/4 adverse effects. Global LINE-1 methylation assessment of serial PBMCs and genome-wide DNA methylation profile in paired tumor biopsies demonstrated minimal changes in global methylation in both regimens. The lack of robust tumor DNA demethylation was accompanied by an absence of the expected 'viral mimicry' inflammatory response, and consequently, no clinical responses were observed. The disease control rate was 7.1%. The median progression-free survival was 1.9 months (95% CI 1.5 to 2.3) and median overall survival was 5 months (95% CI 4.5 to 10). CONCLUSIONS: The evaluated treatment schedules of CC-486 in combination with durvalumab did not demonstrate robust pharmacodynamic or clinical activity in selected immunologically cold solid tumors. Lessons learned from this biomarker-rich study should inform continued drug development efforts using these agents. TRIAL REGISTRATION NUMBER: NCT02811497.

An RB-Condensin II Complex Mediates Long-Range Chromosome Interactions and Influences Expression at Divergently Paired Genes.

Interphase chromosomes are organized into topologically associated domains in order to establish and maintain integrity of transcriptional programs that remain poorly understood. Here, we show that condensin II and TFIIIC are recruited to bidirectionally transcribed promoters by a mechanism that is dependent on the retinoblastoma (RB) protein. Long-range chromosome contacts are disrupted by loss of condensin II loading, which leads to altered expression at bidirectional gene pairs. This study demonstrates that mammalian condensin II functions to organize long-range chromosome contacts and regulate transcription at specific genes. In addition, RB dependence of condensin II suggests that widespread misregulation of chromosome contacts and transcriptional alterations are a consequence of RB mutation.

DNA Methylation as a Robust Classifier of Epithelial Ovarian Cancer.

Epithelial ovarian cancers (EOC) can be defined across different histologic subtypes by specific DNA methylation profiles which correlate with outcome. Identification of distinct EOC methylation subgroups emphasizes the role of epigenetic remodeling to establish unique EOC etiology during transformation, and harbors the potential to direct personalized medicine and patient care.See related article by Bodelon et al., p. 5937.

Deregulation of Retroelements as an Emerging Therapeutic Opportunity in Cancer.

Nearly half of the human genome is comprised of repetitive elements that are tightly regulated to protect the host genome from deleterious consequences associated with their inappropriate activation. Cancer cells often misexpress these elements, in part, due to decreases in DNA methylation. Recent discoveries suggest that tumor suppressor proteins contribute to repression of repetitive elements, and their functional inactivation promotes repeat element misexpression during carcinogenesis. Recent findings also suggest that increased expression of repetitive elements beyond a threshold of tolerance can augment cancer therapy responses. Such advances, reviewed here, paint a picture in which deregulated expression of repetitive genome elements not only contributes to the development of cancer but may also provide a tumor-specific Achilles heel for cancer treatment.

Immunohistochemical Detection of the Retinoblastoma Protein.

The retinoblastoma protein (pRB) plays a key role in proliferative control and genome stability. For these reasons its functions are considered to be tumor suppressive. Its functional status offers critical insight into proliferative control signaling in tissues and in developing malignancies. In this chapter, we outline basic procedures to detect the retinoblastoma protein in formalin fixed, paraffin embedded tissue sections. In addition, we provide protocols to detect phosphorylation levels of pRB in tissues and offer controls to ensure fidelity of measurement. Importantly, these staining methods utilize broadly available reagents and equipment making them accessible to most biomedical research laboratories.

Disruption of CDK-resistant chromatin association by pRB causes DNA damage, mitotic errors, and reduces Condensin II recruitment.

Organization of chromatin structure is indispensible to the maintenance of genome integrity. The retinoblastoma tumor suppressor protein (pRB) mediates both transcriptional repression and chromatin organization, but the independent contributions of these functions have been difficult to study. Here, we utilize a synthetic Rb1 mutant allele (F832A) that maintains pRB association at cell cycle gene promoters, but disrupts a cyclin-dependent kinase (CDK)-resistant interaction with E2F1 to reduce occupancy of pRB on intergenic chromatin. Reduced pRB chromatin association increases spontaneous γH2AX deposition and aneuploidy. Our data indicates that the CDK-resistant pRB-E2F1 scaffold recruits Condensin II to major satellite repeats to stabilize chromatin structure in interphase and mitosis through mechanisms that are distinct from silencing of repetitive sequence expression.

An RB-EZH2 Complex Mediates Silencing of Repetitive DNA Sequences.

Repetitive genomic regions include tandem sequence repeats and interspersed repeats, such as endogenous retroviruses and LINE-1 elements. Repressive heterochromatin domains silence expression of these sequences through mechanisms that remain poorly understood. Here, we present evidence that the retinoblastoma protein (pRB) utilizes a cell-cycle-independent interaction with E2F1 to recruit enhancer of zeste homolog 2 (EZH2) to diverse repeat sequences. These include simple repeats, satellites, LINEs, and endogenous retroviruses as well as transposon fragments. We generated a mutant mouse strain carrying an F832A mutation in Rb1 that is defective for recruitment to repetitive sequences. Loss of pRB-EZH2 complexes from repeats disperses H3K27me3 from these genomic locations and permits repeat expression. Consistent with maintenance of H3K27me3 at the Hox clusters, these mice are developmentally normal. However, susceptibility to lymphoma suggests that pRB-EZH2 recruitment to repetitive elements may be cancer relevant.

Loss of the retinoblastoma tumor suppressor correlates with improved outcome in patients with lung adenocarcinoma treated with surgery and chemotherapy.

The retinoblastoma tumor suppressor pathway is frequently inactivated in human cancer, enabling unrestrained proliferation. Most cancers, however, maintain expression of a wild-type (WT) retinoblastoma tumor suppressor protein (pRB). It is generally in a hyperphosphorylated state (ppRB) because of mutations in upstream regulators such as p16 and cyclin D. Hyperphosphorylated ppRB is considered inactive, although data are emerging that suggest it can retain some function. To test the clinical relevance of pRB status, we obtained archival tissue sections from 91 cases of lung adenocarcinoma resected between 2003 and 2008. All cases received platinum doublet chemotherapy, and the median survival was 5.9 years. All cases were assessed for pRB and ppRB using immunohistochemistry and quantified based on intensity of signal and proportion of positive cells. pRB expression was lost in 15% of lung adenocarcinoma cases. In tumors that did not express pRB, the survival rate was significantly improved (hazard ratio, 0.21; 95% confidence interval, 0.06-0.69; P = .01) in comparison to tumors that express pRB. pRB status was found to be an independent predictor of overall survival on multivariate analysis (hazard ratio, 0.22; 95% confidence interval, 0.07-0.73; P = .01) along with increased stage and age. pRB status did not alter baseline levels of apoptotic or proliferative markers in these tumors, but the DNA damage response protein 53BP1 was higher in cancers with high levels of pRB. In summary, loss of pRB expression is associated with improved survival in patients treated with surgical resection and chemotherapy. This may be useful in classifying patients at greatest benefit for aggressive treatment regimes.

Conditional haploinsufficiency of the retinoblastoma tumor suppressor gene.

Recent work demonstrates that retention of a single functional retinoblastoma susceptibility (RB1) allele is insufficient to maintain genome stability. Haploinsufficiency of RB1 accelerates cancer pathogenesis in concert with inactivation of tumor protein p53. Collectively, multiple lines of evidence suggest revision of the '2-hit' model to include conditional haploinsufficiency of RB1.

Haploinsufficiency of an RB-E2F1-Condensin II complex leads to aberrant replication and aneuploidy.

UNLABELLED: Genome instability is a characteristic of malignant cells; however, evidence for its contribution to tumorigenesis has been enigmatic. In this study, we demonstrate that the retinoblastoma protein, E2F1, and Condensin II localize to discrete genomic locations including major satellite repeats at pericentromeres. In the absence of this complex, aberrant replication ensues followed by defective chromosome segregation in mitosis. Surprisingly, loss of even one copy of the retinoblastoma gene reduced recruitment of Condensin II to pericentromeres and caused this phenotype. Using cancer genome data and gene-targeted mice, we demonstrate that mutation of one copy of RB1 is associated with chromosome copy-number variation in cancer. Our study connects DNA replication and chromosome structure defects with aneuploidy through a dosage-sensitive complex at pericentromeric repeats. SIGNIFICANCE: Genome instability is inherent to most cancers and is the basis for selective killing of cancer cells by genotoxic therapeutics. In this report, we demonstrate that instability can be caused by loss of a single allele of the retinoblastoma gene that prevents proper replication and condensation of pericentromeric chromosomal regions, leading to elevated levels of aneuploidy in cancer.

Loss of the mammalian DREAM complex deregulates chondrocyte proliferation.

Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model that is uniquely deficient for DREAM complex assembly. We have targeted p107 (Rbl1) to prevent MuvB binding and combined it with deficiency for p130 (Rbl2). Our data demonstrate that cells from these mice preferentially assemble BMYB-MuvB complexes and fail to repress transcription. DREAM-deficient mice show defects in endochondral bone formation and die shortly after birth. Micro-computed tomography and histology demonstrate that in the absence of DREAM, chondrocytes fail to arrest proliferation. Since DREAM requires DYRK1A (dual-specificity tyrosine phosphorylation-regulated protein kinase 1A) phosphorylation of LIN52 for assembly, we utilized an embryonic bone culture system and pharmacologic inhibition of (DYRK) kinase to demonstrate a similar defect in endochondral bone growth. This reveals that assembly of mammalian DREAM is required to induce cell cycle exit in chondrocytes.