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.

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.

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.

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.