The overall objective of this investigation is to develop experimentally verified models for circulating fluidized bed (CFB) combustors. Spectral analysis of CFB data obtained at Illinois Institute of Technology shows that the frequencies of pressure oscillations are less than 0.1 Hertz and that they increase with solids volume fraction to the usual value of one Hertz obtained in bubbling beds. These data are consistent with the kinetic theory interpretation of density wave propagation.
Abstract BRCA1 and BRCA2 (Breast cancer susceptibility gene 1 and 2) are tumor suppressor genes that maintain genomic stability by repairing damaged DNA. The proteins coded by these genes are involved in complexes that activate the repair of double strand breaks (DSBs) and initiate homologous recombination (HR). BRCA mutations are common in various forms of human cancers, most notably breast cancer, and previously described in canine tumors. Tumors in canines and humans share many characteristics, including: inter-individual and intra-tumoral heterogeneity, biological behavior, similar tumor microenvironments, and similar genomic mutations. This study interrogates naturally-occuring cancers in dogs as a tool that can add valuable information to our understanding of similar tumors in people. A total of 667 tumors of various histologies were obtained from client-owned dogs and sequenced on the FidoCure® Next-generation sequencing (NGS) panel as part of their veterinary precision medicine platform. Mutations in BRCA1 and BRCA2 were among the 5 most common genetic mutations identified. BRCA1 or BRCA2 mutations were identified in 71.8% (479/667) of tumors overall; 36.4% (n=243) BRCA1 mutations, 33.7% (n=225) BRCA2 mutations, and 1.6% (n=11) BRCA1/2 co-mutations. Germline single-nucleotide polymorphisms (SNPs) represented 56% (136/243) and 61% (138/225) of BRCA1 and BRCA2 mutations, respectively. Removing SNPs and focusing on single-nucleotide variants (SNVs), 16.1% of cases (107/667) had BRCA1 mutations, 13.0% (87/667) had BRCA2 mutations, and 1.65% (11/667) had both BRCA1 and BRCA2 mutations. Utilizing Protein Variation Effect Analyzed (PROVEAN) and Sort Intolerant From Tolerant (SIFT) bioinformatic tools to assess the functional significance of BRCA1 and BRCA2 missense variants, 69.2% and 54.8% of mutations, respectively, were predicted as pathogenic/deleterious. Interestingly, a search of the 2017 MSK IMPACT dataset reveals a pan-cancer prevalence in humans of 2.0% BRCA1 variants (216/10,945 cases) and 3.6% BRCA2 variants (399/10,945 cases). Looking at cancer histologies in dogs, BRCA1 variants were identified in 14.1% of soft tissue sarcomas (STS), 11.4% of apocrine gland anal-sac adenocarcinomas (AGASACA), 11.1% of mast cell tumors (MCT), 10.9% of osteosarcomas (OSA), and 10% of thyroid carcinomas. BRCA2 mutations were identified in 33.3% of MCTs, 32.1% of STSs, 23.8% of histiocytic sarcomas, 17.2% of OSAs, and 16.3% of AGASACAs. This analysis of BRCA1/2 mutations in dogs highlights the value of companion animals as a model for human disease to better understand the contribution of these genes in tumorigenesis and for exploring different targeted therapeutic interventions. Further evaluation of the efficacy of PARP inhibitors, either with or without chemotherapy and/or radiotherapy, in tumors with BRCA1/2 mutations in dogs may reveal information that can then be used to benefit humans with similar malignancies. Citation Format: Benjamin Lewis, Gerry Post, Garrett Harvey, Lindsay Lambert, Aubrey Miller, Rilke Van Buren, Christina Lopes, Lucas Rodrigues. Mutational screening of BRCA1/2 genes in 667 spontaneous canine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2991.
Abstract Cholangiocarcinoma (CCA) is a lethal malignancy of the biliary epithelium that can arise in any part of the biliary tree. Surgery is the only curative treatment for CCA, but only ∼30% of patients present with resectable disease. The remaining 70% of patients present with advanced or metastatic disease and, if eligible, undergo systemic chemotherapy with the first-line combination of gemcitabine and cisplatin. This combination was shown in a phase II clinical trial to significantly increase median survival from 8.1 to 11.7 months, compared to gemcitabine alone. In order to improve on current treatment, pre-clinical evaluation of novel therapeutics is essential to improving outcome. Unfortunately, the paucity of data describing characteristics common to CCA make development of targeted therapy difficult. However, as is true for other types of solid tumors, c-Myc expression likely contributes to CCA phenotype: c-Myc expression has been observed in 95% of CCA tumors, and experimental down-regulation of c-Myc decreases the invasive potential of CCA cells in vitro. Recently it has become possible to inhibit expression of c-Myc using BET inhibitors. Therefore, we evaluated the efficacy of the bromodomain (BET) inhibitor JQ1 using in vivo models of CCA. The five patient-derived xenograft (PDX) models of CCA that we developed are the first such models to be reported. These models retain the heterogeneity, architecture and specific genetic characteristics of the primary tumors from which they were derived. We used three of these models to examine whether the BET inhibitor JQ1 inhibited CCA tumor growth and generated expression profiles of vehicle- and drug-treated tumors. We administered 50 mg/kg of JQ1 i.p. daily for 20 days and monitored tumor growth. This treatment regimen was well tolerated by tumor-bearing mice, without apparent toxicity. Our data demonstrate that JQ1 suppressed tumor growth in two of the three models, compared to vehicle control treated mice. The data also showed that JQ1-treated tumors had lower levels of c-Myc RNA (↓5-fold) and protein and of RNA encoding multiple transcriptional targets downstream of this oncogenic transcription factor. We conclude that BET inhibitors such as JQ1 warrant further investigation as potentially effective drugs for the treatment of CCA. Citation Format: Patrick L. Garcia, Aubrey L. Miller, Kelly Kreitzburg, Tracy L. Gamblin, Leona N. Council, John D. Christein, Pablo Arnoletti, Marty Heslin, Sushanth Reddy, Joseph H. Richardson, Eddy S. Yang, Jun Qi, James E. Bradner, Karina J. Yoon. Bromodomain inhibitor JQ1 inhibits cholangiocarcinoma tumor growth in patient-derived xenograft models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1779. doi:10.1158/1538-7445.AM2015-1779
Gemcitabine is used to treat pancreatic cancer (PC), but is not curative. We sought to determine whether gemcitabine + a BET bromodomain inhibitor was superior to gemcitabine, and identify proteins that may contribute to the efficacy of this combination. This study was based on observations that cell cycle dysregulation and DNA damage augment the efficacy of gemcitabine. BET inhibitors arrest cells in G1 and allow increases in DNA damage, likely due to inhibition of expression of DNA repair proteins Ku80 and RAD51. BET inhibitors (JQ1 or I-BET762) + gemcitabine were synergistic in vitro, in Panc1, MiaPaCa2 and Su86 PC cell lines. JQ1 + gemcitabine was more effective in vivo than either drug alone in patient-derived xenograft models (P < 0.01). Increases in the apoptosis marker cleaved caspase 3 and DNA damage marker γH2AX paralleled antitumor efficacy. Notably, RNA-seq data showed that JQ1 + gemcitabine selectively inhibited HMGCS2 and APOC1 ~6-fold, compared to controls. These proteins contribute to cholesterol biosynthesis and lipid metabolism, and their overexpression supports tumor cell proliferation. IPA data indicated that JQ1 + gemcitabine selectively inhibited the LXR/RXR activation pathway, suggesting the hypothesis that this inhibition may contribute to the observed in vivo efficacy of JQ1 + gemcitabine.
Therapy for rhabdomyosarcoma (RMS) has generally been limited to combinations of conventional cytotoxic agents similar to regimens originally developed in the late 1960s. Recently, identification of molecular alterations through next-generation sequencing of individual tumor specimens has facilitated the use of more targeted therapeutic approaches for various malignancies. Such targeted therapies have revolutionized treatment for some cancer types. However, malignancies common in children, thus far, have been less amenable to such targeted therapies. This report describes the clinical course of an 8-year-old female with embryonal RMS having anaplastic features. This patient experienced multiple relapses after receiving various established and experimental therapies. Genomic testing of this RMS subtype revealed mutations in BCOR, ARID1A, and SETD2 genes, each of which contributes to epigenetic regulation and interacts with or modifies the activity of histone deacetylases (HDAC). Based on these findings, the patient was treated with the HDAC inhibitor vorinostat as a single agent. The tumor responded transiently followed by subsequent disease progression. We also examined the efficacy of vorinostat in a patient-derived xenograft (PDX) model developed using tumor tissue obtained from the patient's most recent tumor resection. The antitumor activity of vorinostat observed with the PDX model reflected clinical observations in that obvious areas of tumor necrosis were evident following exposure to vorinostat. Histologic sections of tumors harvested from PDX tumor-bearing mice treated with vorinostat demonstrated induction of necrosis by this agent. We propose that the evaluation of clinical efficacy in this type of preclinical model merits further evaluation to determine if PDX models predict tumor sensitivity to specific agents and/or combination therapies.
Abstract Cholangiocarcinoma (CCA) is an aggressive bile duct neoplasm which is typically not diagnosed until late stage. The current standard of care, resection followed by gemcitabine with or without cisplatin, is relatively ineffective. Additionally, most patients are ineligible for surgical resection, and postoperative chemotherapy rarely prolongs overall survival. No previously identified mutations (KRAS, SMAD4 and p53) have been shown to drive disease progression. We and others have shown that the proto-oncogene c-Myc is expressed at relatively high levels in CCA cells compared to uninvolved normal tissue, suggesting this oncogene as a prospective therapeutic target in CCA. To evaluate whether c-Myc inhibition affected the proliferation of CCA cells, we evaluated the efficacy of the bromodomain and extraterminal domain (BET) inhibitor JQ1 on c-Myc expression and cell proliferation of CCA cells. JQ1 functions as an acetyl lysine (K-Ac) mimetic which binds to the K-Ac binding pocket of BET protein family members (BRD2, BRD3, BRD4 and BRDT). This binding competitively inhibits the association of BET proteins with K-Ac residues of nuclear proteins and inhibits c-Myc expression. Using a patient-derived xenograft (PDX) model of CCA, we showed previously that JQ1 downregulated c-Myc expression and inhibited tumor growth. Therefore, we hypothesized that JQ1 induces CCA cell death through disruption of BET protein binding to the promoter region of the MYC locus, inducing its downregulation. Our data show that JQ1 decreased c-Myc RNA and protein expression as well as the expression c-Myc transcriptional targets CHK1 and BRCA2. JQ1 also decreased CCA cell viability, clonogenic potential and induced apoptosis of KKU-055 CCA cells in vitro. The likely mechanism of JQ1-mediated c-Myc reduction has been postulated to involve competitive inhibition of the K-Ac binding function of the BET protein BRD4. Our current chromatin immunoprecipitation (ChIP) assays indicate that BRD2 binds to the MYC transcriptional start sites (TSS) P1 and P2, while BRD4 primarily binds MYC TSS P1 in KKU-055 cells. JQ1 precludes these BET proteins from binding their respective sites. When exposed to the IC50 of JQ1, ChIP pulldown with an anti-BRD2 antibody displays a near 100% reduction in binding of BRD2 to the MYC TSS while an anti-BRD4 antibody displays a 70% reduction in binding. In addition, stable BRD2 knockdown models of KKU-055 display reduced c-Myc protein expression by 60 to 80%, while BRD4 knockdown models showed no such reduction. The data demonstrate that in addition to BRD4, BRD2 contributes to regulation of expression of c-Myc in CCA cells. Research funded by NIH/NCI R21 CA205501 Citation Format: Samuel C. Fehling, Aubrey L. Miller, Karina J. Yoon. JQ1 induces cell death and disrupts BET protein binding to the MYC locus in cholangiocarcinoma cells [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 3837.
Abstract Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer (PC), is now the third leading cause of cancer related deaths in the US. This is a highly aggressive disease in that 80% of patients present with locally advanced or metastatic disease and their only treatment option is systemic chemotherapy. Ultimately patient tumors develop resistance to therapy and resulting in a median survival of ~6 months. Therefore, there is an imperative need to identify therapies that provide a more durable response for this patient population. The family of bromodomain and extraterminal domain (BET) proteins has recently become a target of interest for the treatment of PDAC. The BET proteins (BRD2, BRD3, BRD4, and BRDT) function to regulate transcription by recruiting positive transcriptional activators to the promoters of genes. Our lab has shown that inhibition of BET bromodomain function using the BET inhibitor JQ1 suppresses PDAC tumor growth in vivo and inhibits cell viability in vitro. Importantly, PDAC cells and tumors exposed to JQ1 show evidence of DNA damage. We hypothesized that because of the role BET proteins are known to play in regulating gene expression, that the observed JQ1-induced DNA damage may result from JQ1 inhibiting the expression of DNA repair genes whose expression are dependent on BET protein transcriptional complexes. qRT-PCR and immunoblot analysis demonstrated that treatment of the pancreatic cancer cell line BxPC3 with JQ1 inhibited the expression of two double strand break repair proteins, Ku80 and RAD51, both of which have been shown to be overexpressed in cancer including PDAC. Although it has been established that JQ1 co-occupies 99% of the genomic loci that BRD4 is known to bind to, specific gene products whose expression is dependent on BRD4 in PDAC have not been thoroughly investigated. The goal of the current study was to determine if the expression of DNA repair genes Ku80 and RAD51 were specific gene targets of BRD4 transcriptional complexes. We exposed BxPC3 cells to JQ1 for 48 hours and assessed changes in the association of BRD4 with the promoter loci of Ku80 and RAD51 using chromatin immunoprecipitation (ChIP) assays. qRT-PCR of the ChIP demonstrated that treatment with JQ1 significantly (p<0.05) decreased the association of BRD4 with the promoters of both genes. To further confirm that these gene products were dependent on BRD4 for their expression, we down-regulated BRD4 using shRNA and evaluated the effect on the expression of Ku80 and RAD51. Immunoblot revealed that by reducing the expression of BRD4 by 53% in BxPC3 cells, the expression of Ku80 and RAD51 were also decreased by 35% and 93% respectively. We conclude that the expression of Ku80 and RAD51 are dependent on BRD4 transcriptional complexes and are gene targets that contribute to the anti-tumor efficacy of JQ1 in PDAC. Citation Format: Aubrey Lynn Miller, Samuel C. Fehling, Patrick L. Garcia, Karina J. Yoon. BET inhibition decreases the expression of DNA repair enzymes in pancreatic cancer [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 5244.
