Novel BRD2::NUTM1 Fusion in NUT Carcinoma With Exceptional Response to Chemotherapy: A Case Report.

We present the first known case of a patient with BRD2::NUTM1-driven NUT carcinoma. A 59-year-old woman presented with poorly differentiated squamous cell lung cancer metastatic to the pleura. Eventually, a positive NUT immunohistochemistry, NUT fluorescence in situ hybridization, and RNA next-generation sequencing with a BRD2::NUTM1 fusion led to the diagnosis of NUT carcinoma. She received multiple lines of chemotherapy with response and is still alive at 2 years postdiagnosis. This report expands on the known fusions in NUT carcinoma and highlights potential differences in patient prognosis on the basis of gene fusion partners.

Initial Chemotherapy for Locally Advanced and Metastatic NUT Carcinoma.

INTRODUCTION: NUT carcinoma (NC) is an underdiagnosed and aggressive poorly differentiated or squamous cell cancer. A subset of NC is sensitive to chemotherapy, but the optimal regimen is unknown. Experts have recommended platinum- and ifosfamide-based therapy based on case reports. METHODS: Patients with pathologically confirmed NC with known survival outcomes after chemotherapy and consented to participate in a worldwide registry were studied. Results were summarized using descriptive methods. RESULTS: The study included 118 patients with NC. Median age was 34 (range: 1-82) years, 39% were women, and 61% harbored a BRD4::NUTM1 fusion. Patients received platinum (74%) or ifosfamide (26%, including regimens with both, 13%). Of 62 patients with nonmetastatic disease, 40% had a thoracic primary. Compared with platinum-based chemotherapy, patients who received ifosfamide-based chemotherapy had nominally higher progression-free survival (12 mo: 59% [95% CI: 32-87] versus 37% [95% CI: 22-52], hazard ratio = 0.68 [0.32, 1.42], p = 0.3) but not overall survival (OS). Among the 56 patients with metastatic disease, 80% had a thoracic primary. Ifosfamide had an objective response rate (ORR) of 75% (six of eight) and platinum had an ORR of 31% (11 of 36). Nevertheless, there was no difference in progression-free survival or OS. The 3-year OS of the entire cohort was 19% (95% CI: 10%-28%). Of the 11 patients alive greater than 3 years, all presented with nonmetastatic and operable or resectable disease. CONCLUSION: There is a numerically higher ORR for ifosfamide-based therapy compared with platinum-based therapy, with limited durability. OS at 3 years is only 19%, and development of effective therapies is an urgent unmet need for this patient population.

The BRD4-NUT Fusion Alone Drives Malignant Transformation of NUT Carcinoma.

NUT carcinoma (NC) is an aggressive squamous carcinoma defined by the BRD4-NUT fusion oncoprotein. Routinely effective systemic treatments are unavailable for most NC patients. The lack of an adequate animal model precludes identifying and leveraging cell-extrinsic factors therapeutically in NC. Here, we created a genetically engineered mouse model (GEMM) of NC that forms a Brd4::NUTM1 fusion gene upon tamoxifen induction of Sox2-driven Cre. The model displayed complete disease penetrance, with tumors arising from the squamous epithelium weeks after induction and all mice succumbing to the disease shortly thereafter. Closely resembling human NC (hNC), GEMM tumors (mNC) were poorly differentiated squamous carcinomas with high expression of MYC that metastasized to solid organs and regional lymph nodes. Two GEMM-derived cell lines were developed whose transcriptomic and epigenetic landscapes harbored key features of primary GEMM tumors. Importantly, GEMM tumor and cell line transcriptomes co-classified with those of human NC. BRD4-NUT also blocked differentiation and maintained the growth of mNC as in hNC. Mechanistically, GEMM primary tumors and cell lines formed large histone H3K27ac-enriched domains, termed megadomains, that were invariably associated with the expression of key NC-defining proto-oncogenes, Myc and Trp63. Small-molecule BET bromodomain inhibition (BETi) of mNC induced differentiation and growth arrest and prolonged survival of NC GEMMs, as it does in hNC models. Overall, tumor formation in the NC GEMM is definitive evidence that BRD4-NUT alone can potently drive the malignant transformation of squamous progenitor cells into NC. SIGNIFICANCE: The development of an immunocompetent model of NUT carcinoma that closely mimics the human disease provides a valuable global resource for mechanistic and preclinical studies to improve treatment of this incurable disease.

EZH2 Cooperates with BRD4-NUT to Drive NUT Carcinoma Growth by Silencing Key Tumor Suppressor Genes.

NUT carcinoma is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of progrowth genes. BET bromodomain inhibitors (BETi) are a promising treatment for NUT carcinoma that can impede BRD4-NUT's ability to activate genes, but the efficacy of BETi as monotherapy is limited. Here, we demonstrated that enhancer of zeste homolog 2 (EZH2), which silences genes through establishment of repressive chromatin, is a dependency in NUT carcinoma. Inhibition of EZH2 with the clinical compound tazemetostat potently blocked growth of NUT carcinoma cells. Epigenetic and transcriptomic analysis revealed that tazemetostat reversed the EZH2-specific H3K27me3 silencing mark and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4-NUT-regulated genes. Indeed, H3K27me3 and H3K27ac domains were found to be mutually exclusive in NUT carcinoma cells. CDKN2A was identified as the only gene among all tazemetostat-derepressed genes to confer resistance to tazemetostat in a CRISPR-Cas9 screen. Combined inhibition of EZH2 and BET synergized to downregulate cell proliferation genes, resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In preclinical models, combined tazemetostat and BETi synergistically blocked tumor growth and prolonged survival of NUT carcinoma-xenografted mice, with complete remission without relapse in one cohort. Identification of EZH2 as a dependency in NUT carcinoma substantiates the reliance of NUT carcinoma tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary, chromatin regulatory pathways to maintain NUT carcinoma growth. SIGNIFICANCE: Repression of tumor suppressor genes, including CDKN2A, by EZH2 provides a mechanistic rationale for combining EZH2 and BET inhibitors for the clinical treatment of NUT carcinoma. See related commentary by Kazansky and Kentsis, p. 3827.

EZH2 synergizes with BRD4-NUT to drive NUT carcinoma growth through silencing of key tumor suppressor genes.

NUT carcinoma (NC) is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of pro-growth genes. BET bromodomain inhibitors (BETi) impede BRD4-NUT's ability to activate genes and are thus a promising treatment but limited as monotherapy. The role of gene repression in NC is unknown. Here, we demonstrate that EZH2, which silences genes through establishment of repressive chromatin, is a dependency in NC. Inhibition of EZH2 with the clinical compound tazemetostat (taz) potently blocked growth of NC cells. Epigenetic and transcriptomic analysis revealed that taz reversed the EZH2-specific H3K27me3 silencing mark, and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4- NUT-regulated genes. CDKN2A was identified as the only gene amongst all taz-derepressed genes to confer resistance to taz in a CRISPR-Cas9 screen. Combined EZH2 inhibition and BET inhibition synergized to downregulate cell proliferation genes resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In pre-clinical models, combined taz and BETi synergistically blocked growth and prolonged survival of NC-xenografted mice, with all mice cured in one cohort. STATEMENT OF SIGNIFICANCE: Identification of EZH2 as a dependency in NC substantiates the reliance of NC tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary chromatin regulatory pathways to maintain NC growth. In particular, repression of CDKN2A expression by EZH2 provides a mechanistic rationale for combining EZH2i with BETi for the clinical treatment of NC.

Associations between sleep variables and ostensibly paranormal experiences and paranormal beliefs: A scoping review.

Night-time is a period of great significance for many people who report paranormal experiences. However, there is limited understanding of the associations between sleep variables and seemingly paranormal experiences and/or beliefs. The aim of this review is to improve our understanding of these associations while unifying a currently fragmented literature-base into a structured, practical review. In this pre-registered scoping review, we searched for relevant studies in MEDLINE (PubMed), PsycINFO (EBSCO), Web of Science and EMBASE using terms related to sleep and ostensibly paranormal experiences and beliefs. Forty-four studies met all inclusion criteria. All were cross-sectional and most investigated sleep paralysis and/or lucid dreaming in relation to ostensibly paranormal experiences and paranormal beliefs. Overall, there were positive associations between many sleep variables (including sleep paralysis, lucid dreams, nightmares, and hypnagogic hallucinations) and ostensibly paranormal experiences and paranormal beliefs (including those of ghosts, spirits, and near-death experiences). The findings of this review have potential clinical implications such as reducing misdiagnosis and treatment development and provide foundations for further research. Our findings also highlight the importance of understanding why so many people report 'things that go bump in the night'.

Isolated sleep paralysis: Clinical features, perception of aetiology, prevention and disruption strategies in a large international sample.

STUDY OBJECTIVES: Isolated sleep paralysis is a benign but frightening condition characterised by a temporary inability to move at sleep onset or upon awakening. Despite the prevalence of this condition, little is known concerning its clinical features, associated demographic characteristics, and prevention as well as disruption strategies. METHODS: An online cross-sectional study was conducted. The sample comprised 3523 participants who had reported at least one lifetime episode of ISP and 3288 participants without a lifetime episode. Participants answered a survey including questions about sleep quality, sleep paralysis, and sleep paralysis prevention/disruption techniques. RESULTS: A total of 6811 participants were investigated (mean age = 46.9, SD = 15.4, age range = 18-89, 66.1% female). Those who reported experiencing ISP at least once during their lives reported longer sleep onset latencies, shorter sleep duration, and greater insomnia symptoms. Females (vs. male) and younger (vs. older) participants were more likely to experience ISP. Significant fear during episodes was reported by 76.0% of the participants. Most people (63.3%) who experienced ISP believed it to be caused by 'something in the brain'. A minority endorsed supernatural causes (7.1%). Five prevention strategies (e.g., changing sleep position, adjusting sleep patterns) with at least 60.0% effectiveness, and five disruption strategies (e.g., physical/bodily action, making noise) with varying degrees of effectiveness (ranging from 29.5 to 61.8) were identified through open-ended responses. CONCLUSIONS: ISP is associated with shorter sleep duration, longer sleep onset latency, and greater insomnia symptoms. The multiple prevention and disruption techniques identified in this study support existing treatment approaches and may inform subsequent treatment development. Implications for current diagnostic criteria are discussed.

The associations between paranormal beliefs and sleep variables.

Previous studies have found significant associations between paranormal beliefs and sleep variables. However, these have been conducted on a small scale and are limited in the number of sleep variables investigated. This study aims to fill a gap in the literature by investigating paranormal beliefs in relation to a wide range of sleep variables in a large sample. Participants (N = 8853) completed a survey initiated by the BBC Focus Magazine. They reported on their demographics, sleep disturbances and paranormal beliefs. Poorer subjective sleep quality (lower sleep efficiency, longer sleep latency, shorter sleep duration and increased insomnia symptoms) was associated with greater endorsement of belief in: (1) the soul living on after death; (2) the existence of ghosts; (3) demons; (4) an ability for some people to communicate with the dead; (5) near-death experiences are evidence for life after death; and (6) aliens have visited earth. In addition, episodes of exploding head syndrome and isolated sleep paralysis were associated with the belief that aliens have visited earth. Isolated sleep paralysis was also associated with the belief that near-death experiences are evidence for life after death. Findings obtained here indicate that there are associations between beliefs in the paranormal and various sleep variables. This information could potentially better equip us to support sleep via psychoeducation. Mechanisms underlying these associations are likely complex, and need to be further explored to fully understand why people sometimes report "things that go bump in the night".

Case report: Diagnosis of NUT carcinoma of hepatic origin by next-generation sequencing.

NUT carcinoma is a rare subcategory of squamous cell carcinoma. The latter is primarily characterized by the fusion of the coding sequence NUTM1 on chromosome 15q14 with BRD4 or BRD3, both of which are acetyl-histone binding bromodomains. This tumor is often misdiagnosed due to its rarity and its histological similarity with other squamous cell carcinomas. It typically presents as a poorly differentiated squamous cell carcinoma in the head, neck, and mediastinal region, and has no distinct clinical characteristics that set it apart from other malignancies. Although uncommon, other NUT carcinomas have been reported in the literature outside of the midline region. Through next-generation sequencing, we were able to correctly diagnose our patient with the first-documented case of NUT carcinoma of hepatic-only origin.

Stenoparib, an inhibitor of cellular poly (ADP-ribose) polymerases (PARPs), blocks in vitro replication of SARS-CoV-2 variants.

We recently published a preliminary assessment of the activity of a poly (ADP-ribose) polymerase (PARP) inhibitor, stenoparib, also known as 2X-121, which inhibits viral replication by affecting pathways of the host. Here we show that stenoparib effectively inhibits a SARS-CoV-2 wild type (BavPat1/2020) strain and four additional variant strains; alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2) and gamma (P.1) in vitro, with 50% effective concentration (EC50) estimates of 4.1 µM, 8.5 µM, 24.1 µM, 8.2 µM and 13.6 µM, respectively. A separate experiment focusing on a combination of 10 µM stenoparib and 0.5 µM remdesivir, an antiviral drug, resulted in over 80% inhibition of the alpha variant, which is substantially greater than the effect achieved with either drug alone, suggesting at least additive effects from combining the different mechanisms of activity of stenoparib and remdesivir.

NUTM1 -rearranged Carcinoma of the Thyroid : A Distinct Subset of NUT Carcinoma Characterized by Frequent NSD3 - NUTM1 Fusions.

NUT carcinoma (NC) is a rare subtype of squamous cell carcinoma defined by NUTM1 rearrangements encoding NUT fusion oncoproteins (the most frequent fusion partner being BRD4 ) that carries a very poor prognosis, with most patients dying in under 1 year. Only rare primary thyroid NCs have been reported. Here, we evaluated a series of 14 cases. The median patient age at diagnosis was 38 years (range: 17 to 72 y). Eight of 13 cases with slides available for review (62%) showed a morphology typical of NC, whereas 5 (38%) had a non-NC-like morphology, some of which had areas of cribriform or fused follicular architecture resembling a follicular cell-derived thyroid carcinoma. For cases with immunohistochemistry results, 85% (11/13) were positive for NUT on biopsy or resection, though staining was significantly decreased on resection specimens due to fixation; 55% (6/11) were positive for PAX8, and 54% (7/13) for TTF-1. Tumors with a non-NC-like morphology were all positive for PAX8 and TTF-1. The fusion partner was known in 12 cases: 9 (75%) cases had a NSD3-NUTM1 fusion, and 3 (25%) had a BRD4-NUTM1 fusion. For our cohort, the 2-year overall survival (OS) was 69%, and the 5-year OS was 58%. Patients with NC-like tumors had a significantly worse OS compared with that of patients with tumors with a non-NC-like morphology ( P =0.0462). Our study shows that NC of the thyroid can mimic other thyroid primaries, has a high rate of NSD3 - NUTM1 fusions, and an overall more protracted clinical course compared with nonthyroid primary NC.

Efficacy of Oncolytic Herpes Simplex Virus T-VEC Combined with BET Inhibitors as an Innovative Therapy Approach for NUT Carcinoma.

NUT carcinoma (NC) is an extremely aggressive tumor and current treatment regimens offer patients a median survival of six months only. This article reports on the first in vitro studies using immunovirotherapy as a promising therapy option for NC and its feasible combination with BET inhibitors (iBET). Using NC cell lines harboring the BRD4-NUT fusion protein, the cytotoxicity of oncolytic virus talimogene laherparepvec (T-VEC) and the iBET compounds BI894999 and GSK525762 were assessed in vitro in monotherapeutic and combinatorial approaches. Viral replication, marker gene expression, cell proliferation, and IFN-ß dependence of T-VEC efficiency were monitored. T-VEC efficiently infected and replicated in NC cell lines and showed strong cytotoxic effects. This implication could be enhanced by iBET treatment following viral infection. Viral replication was not impaired by iBET treatment. In addition, it was shown that pretreatment of NC cells with IFN-ß does impede the replication as well as the cytotoxicity of T-VEC. T-VEC was found to show great potential for patients suffering from NC. Of note, when applied in combination with iBETs, a reinforcing influence was observed, leading to an even stronger anti-tumor effect. These findings suggest combining virotherapy with diverse molecular therapeutics for the treatment of NC.

NUT carcinoma, an under-recognized malignancy: a clinicopathologic and molecular series of 6 cases showing a subset of patients with better prognosis and a rare ZNF532::NUTM1 fusion.

NUT carcinoma (NC) is a rare malignancy with aggressive clinical behavior, defined by rearrangements involving the NUTM1 gene locus. This entity is often under-recognized and its diagnosis may be challenging. In this study, we describe a subset of patients that, despite the molecularly proven diagnosis of NC, show improved outcomes. In addition, we describe one case with the novel ZNF532::NUTM1 fusion. All cases of NC diagnosed from 2013 to 2022 in our department were retrieved. FISH using dual color bring-together probes and next-generation sequencing assay were performed to characterize the fusions involving NUTM1. Among 6 patients identified, 5 were men with a median age of 35.6 years. Four patients had primary tumors in the head and neck region (2 ethmoid sinus, 1 parotid gland, and 1 lacrimal gland); 1 in the mediastinum, and another presented with a femoral bone tumor. In all cases, the initial diagnoses were not NC. The cases showed different morphological patterns, including monomorphic, rhabdoid, and pleomorphic appearances. One case showed a pseudopapillary pattern. By immunohistochemistry, all tumors showed squamous differentiation and ≥50% of neoplastic cells with nuclear positivity for NUT antibody. One case expressed WT1 (C-terminus) and other showed chromogranin positivity. Genetic study revealed a BRD4::NUTM1 fusion in all head and neck cases, BRD3::NUTM1 in mediastinum case, and ZNF532::NUTM1 fusion in the femur bone case. They were treated with surgical resection plus chemotherapy and radiotherapy. The median overall survival was 23.11 months (1.6-83.3 months) and the median disease-free survival was 14.86 months (0-54.4 months). The patients with longer overall survival were one with a lacrimal gland primary (83.3 months) and other with a parotid lesion (31.9 months). Both patients were primarily treated with complete surgical resection. Anatomic location may be directly related to the overall survival in NC cases. Resectability of the lesion is also an important factor related to survival. Pathologists should include NC in the differential diagnosis of any poorly differentiated and undifferentiated monomorphic malignancy, regardless of its anatomic location.

Serious Cardiac Arrhythmias Detected by Subcutaneous Long-term Cardiac Monitors in Patients With Drug-Resistant Epilepsy.

BACKGROUND AND OBJECTIVES: Epilepsy is associated with an increased risk of cardiovascular disease and premature mortality, including sudden unexpected death in epilepsy (SUDEP). Serious cardiac arrythmias might go undetected in routine epilepsy and cardiac investigations. METHODS: This prospective cohort study aimed to detect cardiac arrhythmias in patients with chronic drug-resistant epilepsy (≥5 years duration) using subcutaneous cardiac monitors for a minimum follow-up duration of 12 months. Participants with known cardiovascular disease or those with abnormal 12-lead ECGs were excluded. The device was programmed to automatically record episodes of tachycardia ≥140 beats per minute (bpm), bradycardia ≤40 bpm for ≥3 seconds, or asystole ≥3 seconds. FINDINGS: Thirty-one patients underwent subcutaneous cardiac monitoring for a median recording duration of 2.2 years (range 0.5-4.2). During this time, 28 patients (90.3%) had episodes of sustained (≥30 seconds) sinus tachycardia, 8/31 (25.8%) had sinus bradycardia, and 3 (9.7%) had asystole. Three patients (9.7%) had serious cardiac arrhythmias requiring additional cardiac interventions. Among them, 2 patients had prolonged sinus arrest and ventricular asystole (>6 seconds), leading to pacemaker insertion in one, and another patient with epileptic encephalopathy had multiple episodes of recurrent nonsustained polymorphic ventricular tachycardia and bundle branch conduction abnormalities. The time to first detection of a clinically significant cardiac arrhythmia ranged between 1.2 and 26.9 months following cardiac monitor insertion. DISCUSSION: Implantable cardiac monitors detected a high incidence of clinically significant cardiac arrhythmias in patients with chronic drug-resistant epilepsy, which may contribute to the incidence of premature mortality, including SUDEP.

Report of the First International Symposium on NUT Carcinoma.

NUT carcinoma is a rare, aggressive cancer defined by rearrangements of the NUTM1 gene. No routinely effective treatments of NUT carcinoma exist, despite harboring a targetable oncoprotein, most commonly BRD4-NUT. The vast majority of cases are fatal. Poor awareness of the disease is a major obstacle to progress in the treatment of NUT carcinoma. While the incidence likely exceeds that of Ewing sarcoma, and BRD4-NUT heralded the bromodomain and extra-terminal domain (BET) inhibitor class of selective epigenetic modulators, NUT carcinoma is incorrectly perceived as "impossibly rare," and therefore receives comparatively little private or governmental funding or prioritization by pharma. To raise awareness, propagate scientific knowledge, and initiate a consensus on standard and targeted treatment of NUT carcinoma, we held the First International Symposium on NUT Carcinoma on March 3, 2021. This virtual event had more than eighty attendees from the Americas, Europe, Asia, and Australia. Patients with NUT carcinoma and family members were represented and shared perspectives. Broadly, the four areas discussed by experts in the field included (1) the biology of NUT carcinoma; (2) standard approaches to the treatment of NUT carcinoma; (3) results of clinical trials using BET inhibitors; and (4) future directions, including novel BET bromodomain inhibitors, combinatorial approaches, and immunotherapy. It was concluded that standard chemotherapeutic approaches and first-generation BET bromodomain inhibitors, the latter complicated by a narrow therapeutic window, are only modestly effective in a minority of cases. Nonetheless, emerging second-generation targeted inhibitors, novel rational synergistic combinations, and the incorporation of immuno-oncology approaches hold promise to improve the prognosis of this disease.

Reprogrammed tracrRNAs enable repurposing of RNAs as crRNAs and sequence-specific RNA biosensors.

In type II CRISPR systems, the guide RNA (gRNA) comprises a CRISPR RNA (crRNA) and a hybridized trans-acting CRISPR RNA (tracrRNA), both being essential in guided DNA targeting functions. Although tracrRNAs are diverse in sequence and structure across type II CRISPR systems, the programmability of crRNA-tracrRNA hybridization for Cas9 is not fully understood. Here, we reveal the programmability of crRNA-tracrRNA hybridization for Streptococcus pyogenes Cas9, and in doing so, redefine the capabilities of Cas9 proteins and the sources of crRNAs, providing new biosensing applications for type II CRISPR systems. By reprogramming the crRNA-tracrRNA hybridized sequence, we show that engineered crRNA-tracrRNA interactions can not only enable the design of orthogonal cellular computing devices but also facilitate the hijacking of endogenous small RNAs/mRNAs as crRNAs. We subsequently describe how these re-engineered gRNA pairings can be implemented as RNA sensors, capable of monitoring the transcriptional activity of various environment-responsive genomic genes, or detecting SARS-CoV-2 RNA in vitro, as an Atypical gRNA-activated Transcription Halting Alarm (AGATHA) biosensor.

Dr James Ost's contributions to the work of the British false memory society.

The British False Memory Society (BFMS) is a registered charity founded in 1993 in response to an epidemic of false claims of past childhood sexual abuse by adults in therapy. The accusers believe they have recovered unconscious memories of a hidden past, but scientific and other evidence raise the possibility of false memories or retrospective reappraisal. The BFMS aims to raise awareness about false memory and to reduce the impact of the resulting false accusations. Dr James Ost was an active member of the BFMS's Scientific and Professional Advisory Board. Three lines of his research were particularly relevant to the work of the BFMS. The first of these was his investigations of retractors. His insights provided a deeper understanding of processes involved in the formation and subsequent rejection of false memories and beliefs relating to such allegations. He also carried out experimental studies providing empirical proof that false memories can be implanted under well controlled conditions. Finally, he carried out, and produced reviews of, surveys of misconceptions about the nature of memory, thus highlighting issues that have major implications for the working of the legal system. Dr Ost also served as an expert defence witness on a number of occasions.