Citations

Abstract Background: Epigenetic dysregulation is involved in the etiology and progression of various human diseases. Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology samples, and thus FFPE samples are a major resource of samples in clinical research. However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples and are hampered by low chromatin yield in FFPE samples due to the lack of reliable and efficient chromatin preparation methods. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays. Results: During rehydration of FFPE tissues, applying tissue-level cross-link reversal to the deparaffinized tissue at 65°C dramatically increased chromatin yield in the soluble fraction. We found the resulting chromatin to be compatible with ChIP-qPCR and ChIP-seq analysis of histone marks (H3K4me3, H3K27me3, and H3K27Ac) and RNA polymerase II. The chromatin prepared by Chrom-EX PE showed a gradual fragmentation pattern with varying incubation temperature. At temperatures below 37°C, the majority of soluble chromatin was over 1 kb in size. The soluble chromatin prepared at 45-60°C showed a typical nucleosomal pattern shown in cell lines and frozen tissues. The majority of the chromatin prepared at 65°C is close to mononucleosomal size. These observations indicate that chromatin preparation from FFPE samples can be controlled for downstream chromatin-based epigenetic assays. Conclusions: Our method achieves efficient extraction of high-quality chromatin from FFPE samples suitable for targeted and genome-wide epigenetic assays while minimizing degradation and damages. This approach provides a new platform for developing chromatin-based assays applicable to archived FFPE samples to circumvent the over-fixed nature of FFPE tissues. This work was supported, in part, by National Institutes of Health grant P01DK068055 and the Mayo Clinic Center for Individualized Medicine Epigenomics Program. Citation Format: Jian Zhong, Zhenqing Ye, Chad Clark, Samuel Lenz, Justin Nguyen, Huihuang Yan, Keith Robertson, Gianrico Farrugia, Zhiguo Zhang, Tamas Ordog, Jeong-Heon Lee. Enhanced and controlled chromatin extraction for chromatin-based epigenetic assays in FFPE tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5180.

Abstract Fibroproliferative diseases are a leading cause of morbidity and mortality featuring localized and systematic tissue/organ fibrosis, which account for nearly 45% of all deaths in the developed world, with an unknown etiology and no effective treatment. Transforming growth factor beta (TGF-β) is a ubiquitous cytokine that contributes to fibroblast activation, myofibroblast differentiation, collagen overproduction and pathological tissue fibrosis. In that mounting evidence indicate that altered glutamine metabolism has critical roles in regulating signaling pathways, maintaining redox balance and macromolecular biosynthesis, we wished to test the general hypothesis that intervention of these metabolic processes could provide novel approaches to developing anti-fibrotic therapies. To that end, in the current work we provide a framework integrating glutamine dysregulation with profibrotic TGF-β signaling. We found that Glutaminase 1 (GLS1), which converts glutamine to glutamate, is induced by TGF-β and is also elevated in (i) fibroblasts isolated from Idiopathic Pulmonary Fibrosis (IPF) patients; and (ii) the murine bleomycin (i.e., TGF-β driven) model of lung fibrosis. While phosphorylation of Smad2 and Smad3 occurs independently of GLS1, TGF-β stimulated expression of profibrotic targets (i.e., Collagen I, PAI-1, CTGF, α-SMA and fibronectin) as well as biological actions (i.e., cell migration and anchorage independent growth) require GLS1 activity. Moreover, knockdown of Smad2 or Smad3 as well as inhibition of MAPK, PI3K, Akt or mTOR abrogates the induction of GLS1 by TGF-β; documenting that TGF-β regulates GLS1 expression via both Smad and non-Smad signaling. As GLS1 represents a critical component mediating the fibroproliferative actions of TGF-β, in order to identify the operative molecular mechanism(s) we focused on (i) a group of histone deacetylases called sirtuins (SIRT) which are known to elicit antifibrotic effects; and (ii) the Forkhead box protein O4 (FOXO4) transcription factor which has been shown to bind the GLS1 promoter. We found that there is not only a decrease in SIRT7 and FOXO4 coincident with the induction of GLS1 by TGF-β, but TGF-β inhibits SIRT7 deacetylation activity which in turn prevents FOXO4 deacetylation and inhibition of GLS1 expression. Last, consistent with SIRT7 and FOXO4 being negative regulators of profibrotic TGF-β signaling, their levels were also decreased in IPF fibroblasts and bleomycin-induced lung fibrosis. These studies point to an exciting, novel and unexplored connection between sirtuins, FOXOs and glutamine metabolism by elucidating the mechanism(s) by which epigenetic and transcriptional processes cooperate to regulate glutamine metabolism and fibrotic development in a TGF-β dependent manner. Citation Format: Malay Choudhury, Xueqian Yin, Jeong-Han Kang, Mahefatiana Andrianifahanana, Edward B. Leof. Cooperativity between glutaminase 1 and histone deacetylase SIRT7 regulates TGF-β mediated lung fibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1847.

AbstractAnaplastic large cell lymphomas (ALCLs) represent a relatively common group of T-cell non-Hodgkin lymphomas (T-NHLs) that are unified by similar pathologic features but demonstrate marked genetic heterogeneity. ALCLs are broadly classified as being anaplastic lymphoma kinase (ALK)+ or ALK−, based on the presence or absence of ALK rearrangements. Exome sequencing of 62 T-NHLs identified a previously unreported recurrent mutation in the musculin gene, MSCE116K, exclusively in ALK− ALCLs. Additional sequencing for a total of 238 T-NHLs confirmed the specificity of MSCE116K for ALK− ALCL and further demonstrated that 14 of 15 mutated cases (93%) had coexisting DUSP22 rearrangements. Musculin is a basic helix-loop-helix (bHLH) transcription factor that heterodimerizes with other bHLH proteins to regulate lymphocyte development. The E116K mutation localized to the DNA binding domain of musculin and permitted formation of musculin–bHLH heterodimers but prevented their binding to authentic target sequence. Functional analysis showed MSCE116K acted in a dominant-negative fashion, reversing wild-type musculin-induced repression of MYC and cell cycle inhibition. Chromatin immunoprecipitation–sequencing and transcriptome analysis identified the cell cycle regulatory gene E2F2 as a direct transcriptional target of musculin. MSCE116K reversed E2F2-induced cell cycle arrest and promoted expression of the CD30–IRF4–MYC axis, whereas its expression was reciprocally induced by binding of IRF4 to the MSC promoter. Finally, ALCL cells expressing MSCE116K were preferentially targeted by the BET inhibitor JQ1. These findings identify a novel recurrent MSC mutation as a key driver of the CD30–IRF4–MYC axis and cell cycle progression in a unique subset of ALCLs.

Abstract Aims Left atrial (LA) function is impaired in heart failure with preserved ejection fraction (HFpEF) and predicts disease severity and risk of adverse outcome. We sought to evaluate whether LA strain could enhance diagnosis of HFpEF. Methods and results Consecutive patients with unexplained exertional dyspnoea and image quality suitable for measuring LA strain underwent invasive cardiopulmonary exercise testing to ascertain the presence of HFpEF or non-cardiac causes of dyspnoea (NCD). LA reservoir strain was measured in all patients, while LA conduit and booster strain were measured in patients with sinus rhythm. LA strain was measurable in 363 of 378 examinations (96.5%; HFpEF: 238, NCD: 125). LA reservoir strain (29 ± 16% vs. 40 ± 13%, P < 0.0001) and conduit strain (18 ± 10% vs. 22 ± 10%, P = 0.0001) were significantly impaired in HFpEF compared to NCD. Of all echocardiographic indices, LA reservoir strain best discriminated HFpEF from NCD [area under the curve (AUC) 0.719, P < 0.0001], outperforming E/e' (AUC difference +0.117, P < 0.0001), LA enlargement (+0.090, P = 0.001), tricuspid regurgitation velocity > 2.8 m/s (+0.082, P = 0.0085), left ventricular hypertrophy (+0.0159, P < 0.0001) and left ventricular global longitudinal strain (+0.0198, P < 0.0001). Indexing LA reservoir strain to estimated LA pressure (E/e') as a surrogate for LA compliance further improved diagnostic performance (AUC 0.772, P < 0.0001; +0.053 compared to LA reservoir strain alone, P = 0.003). Conclusion Left atrial reservoir strain may provide enhanced diagnostic accuracy beyond conventional echocardiographic measures to discriminate HFpEF from NCD. Further study is required to verify the diagnostic utility of LA strain.

Background Clinical investigations of shock in cardiac intensive care units (CICUs) have primarily focused on acute myocardial infarction (AMI) complicated by cardiogenic shock (AMICS). Few studies have evaluated the full spectrum of shock in contemporary CICUs. Methods and Results The Critical Care Cardiology Trials Network is a multicenter network of advanced CICUs in North America. Anytime between September 2017 and September 2018, each center (n=16) contributed a 2-month snap-shot of all consecutive medical admissions to the CICU. Data were submitted to the central coordinating center (TIMI Study Group, Boston, MA). Shock was defined as sustained systolic blood pressure <90 mm Hg with end-organ dysfunction ascribed to the hypotension. Shock type was classified by site investigators as cardiogenic, distributive, hypovolemic, or mixed. Among 3049 CICU admissions, 677 (22%) met clinical criteria for shock. Shock type was varied, with 66% assessed as cardiogenic shock (CS), 7% as distributive, 3% as hypovolemic, 20% as mixed, and 4% as unknown. Among patients with CS (n=450), 30% had AMICS, 18% had ischemic cardiomyopathy without AMI, 28% had nonischemic cardiomyopathy, and 17% had a cardiac cause other than primary myocardial dysfunction. Patients with mixed shock had cardiovascular comorbidities similar to patients with CS. The median CICU stay was 4.0 days (interquartile range [IQR], 2.5–8.1 days) for AMICS, 4.3 days (IQR, 2.1–8.5 days) for CS not related to AMI, and 5.8 days (IQR, 2.9–10.0 days) for mixed shock versus 1.9 days (IQR, 1.0–3.6) for patients without shock ( P <0.01 for each). Median Sequential Organ Failure Assessment scores were higher in patients with mixed shock (10; IQR, 6–13) versus AMICS (8; IQR, 5–11) or CS without AMI (7; IQR, 5–11; each P <0.01). In-hospital mortality rates were 36% (95% CI, 28%–45%), 31% (95% CI, 26%–36%), and 39% (95% CI, 31%–48%) in AMICS, CS without AMI, and mixed shock, respectively. Conclusions The epidemiology of shock in contemporary advanced CICUs is varied, and AMICS now represents less than one-third of all CS. Despite advanced therapies, mortality in CS and mixed shock remains high. Investigation of management strategies and new therapies to treat shock in the CICU should take this epidemiology into account.

CD4 and CD8 T cells are vital components of the immune system. We found that histone deacetylase 3 (HDAC3) is critical for the development of CD4 T cells, as HDAC3-deficient DP thymocytes generate only CD8SP thymocytes in mice. In the absence of HDAC3, MHC Class II-restricted OT-II thymocytes are redirected to the CD8 cytotoxic lineage, which occurs with accelerated kinetics. Analysis of histone acetylation and RNA-seq reveals that HDAC3-deficient DP thymocytes are biased towards the CD8 lineage prior to positive selection. Commitment to the CD4 or CD8 lineage is determined by whether persistent TCR signaling or cytokine signaling predominates, respectively. Despite elevated IL-21R/γc/STAT5 signaling in HDAC3-deficient DP thymocytes, blocking IL-21R does not restore CD4 lineage commitment. Instead, HDAC3 binds directly to CD8-lineage promoting genes. Thus, HDAC3 is required to restrain CD8-lineage genes in DP thymocytes for the generation of CD4 T cells.