NUT midline carcinoma (NMC), a subtype of squamous cell cancer, is one of the most aggressive human solid malignancies known. NMC is driven by the creation of a translocation oncoprotein, BRD4-NUT, ...which blocks differentiation and drives growth of NMC cells. BRD4-NUT forms distinctive nuclear foci in patient tumors, which we found correlate with ∼100 unprecedented, hyperacetylated expanses of chromatin that reach up to 2 Mb in size. These "megadomains" appear to be the result of aberrant, feed-forward loops of acetylation and binding of acetylated histones that drive transcription of underlying DNA in NMC patient cells and naïve cells induced to express BRD4-NUT. Megadomain locations are typically cell lineage-specific; however, the cMYC and TP63 regions are targeted in all NMCs tested and play functional roles in tumor growth. Megadomains appear to originate from select pre-existing enhancers that progressively broaden but are ultimately delimited by topologically associating domain (TAD) boundaries. Therefore, our findings establish a basis for understanding the powerful role played by large-scale chromatin organization in normal and aberrant lineage-specific gene transcription.
To investigate the mechanism that drives dramatic mistargeting of active chromatin in NUT midline carcinoma (NMC), we have identified protein interactions unique to the BRD4–NUT fusion oncoprotein ...compared with wild-type BRD4. Using cross-linking, affinity purification, and mass spectrometry, we identified the EP300 acetyltransferase as uniquely associated with BRD4 through the NUT fusion in both NMC and non-NMC cell types. We also discovered ZNF532 associated with BRD4–NUT in NMC patient cells but not detectable in 293T cells. EP300 and ZNF532 are both implicated in feed-forward regulatory loops leading to propagation of the oncogenic chromatin complex in BRD4–NUT patient cells. Adding key functional significance to our biochemical findings, we independently discovered a ZNF532–NUT translocation fusion in a newly diagnosed NMC patient. ChIP sequencing of the major players NUT, ZNF532, BRD4, EP300, and H3K27ac revealed the formation of ZNF532–NUT–associated hyperacetylated megadomains, distinctly localized but otherwise analogous to those found in BRD4–NUT patient cells. Our results support a model in which NMC is dependent on ectopic NUT-mediated interactions between EP300 and components of BRD4 regulatory complexes, leading to a cascade of misregulation.
NUT midline carcinoma (NMC) is an aggressive subtype of squamous cell carcinoma that typically harbors BRD4/3-NUT fusion oncoproteins that block differentiation and maintain tumor growth. In 20% of ...cases, NUT is fused to uncharacterized non-BRD gene(s). We established a new patient-derived NMC cell line (1221) and demonstrated that it harbors a novel NSD3-NUT fusion oncogene. We find that NSD3-NUT is both necessary and sufficient for the blockade of differentiation and maintenance of proliferation in NMC cells. NSD3-NUT binds to BRD4, and BRD bromodomain inhibitors induce differentiation and arrest proliferation of 1221 cells. We find further that NSD3 is required for the blockade of differentiation in BRD4-NUT-expressing NMCs. These findings identify NSD3 as a novel critical oncogenic component and potential therapeutic target in NMC.
The existence of a family of fusion oncogenes in squamous cell carcinoma is unprecedented, and should lead to key insights into aberrant differentiation in NMC and possibly other squamous cell carcinomas. The involvement of the NSD3 methyltransferase as a component of the NUT fusion protein oncogenic complex identifies a new potential therapeutic target.
NUT midline carcinoma (NMC) is an aggressive type of squamous cell carcinoma that is defined by the presence of BRD-NUT fusion oncogenes, which encode chimeric proteins that block differentiation and ...maintain tumor growth. BRD-NUT oncoproteins contain two bromodomains whose binding to acetylated histones is required for the blockade of differentiation in NMC, but the mechanisms by which BRD-NUT act remain uncertain. Here, we provide evidence that MYC is a key downstream target of BRD4-NUT. Expression profiling of NMCs shows that the set of genes whose expression is maintained by BRD4-NUT is highly enriched for MYC upregulated genes, and MYC and BRD4-NUT protein expression is strongly correlated in primary NMCs. More directly, we find that BRD4-NUT associates with the MYC promoter and is required to maintain MYC expression in NMC cell lines. Moreover, both siRNA knockdown of MYC and a dominant-negative form of MYC, omomyc, induce differentiation of NMC cells. Conversely, differentiation of NMC cells induced by knockdown of BRD4-NUT is abrogated by enforced expression of MYC. Together, these findings suggest that MYC is a downstream target of BRD4-NUT that is required for maintenance of NMC cells in an undifferentiated, proliferative state. Our findings support a model in which dysregulation of MYC by BRD-NUT fusion proteins has a central role in the pathogenesis of NMC.
•Blue LED light has similar cytotoxicity to mammalian cells as Blue fluorescent light.•Blue LED light is genotoxic but less than Blue florescent light.•DNA repair deficient cells are sensitive to ...Blue LED light exposure.•Antioxidant, ascorbic acid 2 glucoside can protect cells from blue LED light induced cytotoxicity.
Light emitting diode (LED) devices emit narrow bands of the blue, green, and red light spectrum rather than the continuous spectrum emitted from sunlight and fluorescent light bulbs. LED devices have become considerably common in society, and the fluence of blue light from LED devices is more intense than other light sources. Previous studies presented that the blue light spectrum may harness potentially inimical genotoxicity. Therefore, the aim of this study was to investigate this potential cytotoxicity and genotoxicity, as well as identify the mechanism of the cellular effects induced by blue LED light exposure in mammalian cell lines with their DNA repair deficient mutants. Our results demonstrated that blue LED light induced both oxidative stress to cells and cytotoxic and genotoxic effects including reduction of clonogenicity, cell cycle arrest, induction of sister chromatid exchanges, endoreduplicated chromosomes, and increased frequency of HPRT locus mutations. In DNA repair deficient cells, particularly those involving double strand break repair deficiency, cells presented hypersensitivity to blue LED light exposure. Blue LED light also induced chromosome aberrations more in DNA repair deficient cells than wild type cells. The cytotoxicity of blue LED light was reduced by an effective antioxidant, ascorbic acid 2-glucoside, which can suppress blue LED light induced oxidative stress. These results indicated that prolonged, high intensity exposure to blue LED light induces genotoxic stress to cells, and oxidative stress induced by blue LED light is targeting DNA to induce these biological effects.
A quality improvement initiative (QII) was conducted with five community-based health systems' oncology care centers (sites A–E). The QII aimed to increase referrals, genetic counseling (GC), and ...germline genetic testing (GT) for patients with ovarian cancer (OC) and triple-negative breast cancer (TNBC).
QII activities occurred at sites over several years, all concluding by December 2020. Medical records of patients with OC and TNBC were reviewed, and rates of referral, GC, and GT of patients diagnosed during the 2 years before the QII were compared to those diagnosed during the QII. Outcomes were analyzed using descriptive statistics, two-sample t-test, chi-squared/Fisher's exact test, and logistic regression.
For patients with OC, improvement was observed in the rate of referral (from 70% to 79%), GC (from 44% to 61%), GT (from 54% to 62%) and decreased time from diagnosis to GC and GT. For patients with TNBC, increased rates of referral (from 90% to 92%), GC (from 68% to 72%) and GT (81% to 86%) were observed. Effective interventions streamlined GC scheduling and standardized referral processes.
A multi-year QII increased patient referral and uptake of recommended genetics services across five unique community-based oncology care settings.
•A coordinated quality improvement initiative was completed in five community-based health system oncology care centers.•Referral, genetic counseling, and genetic testing improved for patients with ovarian and triple-negative breast cancer.•Average time to completion of genetic testing for patients with ovarian cancer significantly decreased at almost all sites.•The most effective quality improvement interventions standardized genetic counseling referral and delivery processes.
The University of Texas MD Anderson Cancer Center, a comprehensive cancer center designated by the National Cancer Institute (NCI), defines its service population area as the State of Texas (29.1 M), ...the second most populous state in the country and the state with the greatest number of uninsured residents in the United States. Consistent with a novel and formal commitment to prevention as part of its core mission, alongside clear opportunities in Texas to drive vaccine uptake, MD Anderson assembled a transdisciplinary team to develop an institutional Framework to increase adolescent HPV vaccination and reduce HPV-related cancer burden. The Framework was developed and activated through a four-phase approach aligned with the NCI Cancer Center Support Grant Community Outreach and Engagement component. MD Anderson identified collaborators through data-driven outreach and constructed a portfolio of collaborative multi-sector initiatives through review processes designed to assess readiness, impact and sustainability. The result is an implementation community of 78 institutions collaboratively implementing 12 initiatives within a shared measurement framework impacting 18 counties. This paper describes a structured and rigorous process to set up the implementation of a multi-year investment in evidence-based strategies to increase HPV vaccination that solves challenges preventing implementation of recommended strategies and to encourage similar initiative replication.
Program evaluation can identify the successes and challenges of implementing clinical programs, which can inform future dissemination efforts. A cancer genetics improvement program, disseminated from ...the Lead Team's institution to five health systems (Participating Sites), was genetic counselor led, using virtual implementation facilitation to support Participating Sites' performance of quality improvement (QI) activities over several years. Program implementation and outcome evaluations were performed and included evaluation of program delivery and initial effects of the program on Participating Sites. A logic model guided evaluation of program implementation (inputs, activities, outputs, delivery/fidelity, and coverage/reach) and initial outcomes (short‐term and intermediate outcomes). Data were collected from program documents and an Evaluation Survey of Participating Site team members (21 respondents), compared against the Lead Team's expectations of participation, and analyzed using descriptive statistics. All program inputs, outputs, and activities were available and delivered as expected across the five Participating Sites. The most frequently used activities and inputs were facilitation‐associated meetings and meeting resources, which were rated as useful/helpful by the majority of respondents. Nearly all respondents noted improvement in short‐term outcomes following participation: 82.4% reported increased awareness of clinical processes, 94.1% increased knowledge of QI methods, 100% reported increased perceived importance of QI, 94.1% increased perceived feasibility of QI, and 76.5% reported increased problem‐solving skills and self‐efficacy to use QI at their site. Intermediate outcomes (identifying barriers, developing interventions, improved teamwork, and capacity) were achieved following program participation as indicated by the results of the program document review and Evaluation Survey responses. Implementation challenges at Participating Sites included staffing constraints, difficulties obtaining buy‐in and participation, and developing interventions over time. The multi‐site improvement program was delivered and implemented with high levels of fidelity and resulted in improved short and intermediate outcomes. Future research will evaluate long‐term, patient‐level outcomes associated with site‐specific QI interventions.
The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and ...targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation.
•Nutlin-3 inhibits myoblast proliferation and prevents differentiation into myotubes.•Nutlin-3 increases MDM2 expression and down-regulates Rb protein levels.•This study has implication in nutlin-3 treatment of rhabdomyosarcomas.
PDF
