EZH2 Cooperates with BRD4-NUT to Drive NUT Carcinoma Growth by Silencing 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) are a promising treatment for NC that can impede BRD4-NUT’s ability to activate genes, but the efficacy of BETi as monotherapy are limited. Here, we demonstrated 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. Indeed, H3K27me3 and H3K27ac domains were found to be mutually exclusive in NC cells. CDKN2A was identified as the only gene among all taz-derepressed genes to confer resistance to taz 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 pre-clinical models, combined taz and BETi synergistically blocked tumor growth and prolonged survival of NC-xenografted mice, with complete remission without relapse in one cohort. 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

Trabecular bone score is associated with volumetric bone density and microarchitecture as assessed by central QCT and HRpQCT in Chinese American and white women.

Although high-resolution peripheral quantitative computed tomography (HRpQCT) and central quantitative computed tomography (QCT) studies have shown bone structural differences between Chinese American (CH) and white (WH) women, these techniques are not readily available in the clinical setting. The trabecular bone score (TBS) estimates trabecular microarchitecture from dual-energy X-ray absorptiometry spine images. We assessed TBS in CH and WH women and investigated whether TBS is associated with QCT and HRpQCT indices. Areal bone mineral density (aBMD) by dual-energy X-ray absorptiometry, lumbar spine (LS) TBS, QCT of the LS and hip, and HRpQCT of the radius and tibia were performed in 71 pre- (37 WH and 34 CH) and 44 postmenopausal (21 WH and 23 CH) women. TBS did not differ by race in either pre- or postmenopausal women. In the entire cohort, TBS positively correlated with LS trabecular volumetric bone mineral density (vBMD) (r = 0.664), femoral neck integral (r = 0.651), trabecular (r = 0.641) and cortical vBMD (r = 0.346), and cortical thickness (C/I; r = 0.540) by QCT (p < 0.001 for all). TBS also correlated with integral (r = 0.643), trabecular (r = 0.574) and cortical vBMD (r = 0.491), and C/I (r = 0.541) at the total hip (p < 0.001 for all). The combination of TBS and LS aBMD predicted more of the variance in QCT measures than aBMD alone. TBS was associated with all HRpQCT indices (r = 0.20-0.52) except radial cortical thickness and tibial trabecular thickness. Significant associations between TBS and measures of HRpQCT and QCT in WH and CH pre- and postmenopausal women demonstrated here suggest that TBS may be a useful adjunct to aBMD for assessing bone quality

High Dose Hematopoietic Stem Cell Transplantation Leads to Rapid Hematopoietic and Microglia Recovery and Disease Correction in a Mouse Model of Hurler Syndrome

Background . Allogeneic hematopoietic stem cell transplant (HSCT) is a promising approach to halt disease progression and prevent or ameliorate neurological symptoms arising from select inherited metabolic disorders (IMDs). Donor-derived cells, including microglia, limit disease progression post-HSCT via production of normal enzyme in a process called cross-correction. A standard cell dose used in HSCT is sub-optimal, resulting in delayed hematopoietic recovery and slower correction of central nervous system (CNS) defects (Lund et al BBMT 2019). To address these limitations, we developed MGTA-456, a cell therapy that contains large numbers of CD34+ cells and has led to accelerated neutrophil recovery and 100% engraftment post-HSCT in patients with malignant and non-malignant diseases (Wagner et al Blood 2017; Orchard et al AAN 2019). We previously showed that MGTA-456 leads to faster hematopoietic and microglia recovery in the brains of NSG mice (Goncalves et al AAN 2019); however, the impact of cell dose on disease outcomes and mechanism of cross-correction are unknown. Here, we show that faster and greater hematopoietic and microglia recovery leads to rapid and complete resolution of disease endpoints in a mouse model of mucopolysaccharidosis I (Hurler syndrome) and that, mechanistically, donor engraftment in the brain is required for disease cross-correction. Results . To determine whether cell dose impacts microglial engraftment, CD45.2 mice were conditioned with a clinically-relevant, myeloablative dose of busulfan and transplanted with increasing doses of CD45.1 bone marrow cells, beginning with 0.3x106 cells/mouse (2x106 cells/kg) based on allometric scaling to model high dose cell therapies. A dose-dependent increase in microglia was observed as early as 1 week post-HSCT, where 10x106 cells led to a 26-fold higher number of donor microglia compared to 0.3x106 cells (p&lt;0.01), an effect that was sustained through 16 weeks post-HSCT (p&lt;0.001). Despite high donor chimerism in the periphery at all cell doses (75-99%), only partial chimerism was observed in the brain. At 16 weeks, donor microglia represented only 2% of microglia after transplant of 0.3x106 cells but this was increased to 35% of total microglia in the brain following transplant of 10x106 cells. These data indicate that while busulfan can facilitate a low level of microglia engraftment, this effect can be enhanced by transplant of high cell doses. To evaluate the impact of cell dose on disease outcomes, we transplanted a low (0.3x106) or high (10x106) cell dose of wild-type bone marrow cells into busulfan-conditioned Idua-/- mice, a model of Hurler syndrome. At 1 month post-HSCT, peripheral donor myeloid chimerism was &gt;75% and &gt;99% for 0.3x106 and 10x106 cells, respectively. In the brain, transplant of 10x106 cells led to significantly higher donor microglial engraftment versus 0.3x106 cells (Figure A). Notably, high cell dose resulted in significantly higher levels of IDUA enzyme in the brain (Figure B), reduced levels of β-hexosaminidase and glycosaminoglycan (GAG) substrate, and normalization of behavioral outcomes, including rotarod performance, to wild type levels (Figure C). In peripheral tissues, transplant of 10x106 cells, but not 0.3x106 cells, led to a reduction of GAGs to wild type levels as early as 1 week post-HSCT (p&lt;0.01). To determine if donor engraftment in the brain is required for cross-correction, we transplanted 10x106 cells into mice conditioned with a myeloablative dose of treosulfan, which is not sufficient to condition the brain for microglia engraftment. Treosulfan conditioning, followed by high dose HSCT, led to &gt;99% donor myeloid chimerism in the periphery but neither increased microglial levels nor corrected CNS defects (Figures A-C), suggesting that donor engraftment in the brain is required for disease modification. Long-term outcomes and impact on skeletal phenotype in this model will also be presented. Conclusions . We demonstrate that high dose HSCT leads to robust microglia engraftment in the brain and improved disease endpoints. These data suggest that strategies to increase cell dose, such as MGTA-456, may accelerate resolution of neurologic disease in patients with IMDs. Similar approaches, possibly coupled with gene modification technologies, could be used to improve microglial function in other neurodegenerative diseases where defective microglia have been implicated. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership, Patents &amp; Royalties. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents &amp; Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents &amp; Royalties. </jats:sec

Supplementary Fig. S4 from EZH2 Cooperates with BRD4-NUT to Drive NUT Carcinoma Growth by Silencing Key Tumor Suppressor Genes

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Supplementary Tables S5-8 from EZH2 Cooperates with BRD4-NUT to Drive NUT Carcinoma Growth by Silencing Key Tumor Suppressor Genes

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Supplementary Fig. S1 from EZH2 Cooperates with BRD4-NUT to Drive NUT Carcinoma Growth by Silencing Key Tumor Suppressor Genes

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