Neuroblastoma is a pediatric tumor characterized by histologic heterogeneity, and accounts for ~15% of childhood deaths from cancer. The five-year survival for patients with high-risk stage 4 disease has not improved in two decades. We used whole exome sequencing (WES) to identify mutations present in three independent high-risk stage 4 neuroblastoma tumors (COA/UAB-3, COA/UAB -6 and COA/UAB -8) and a stage 3 tumor (COA/UAB-14). Among the four tumors WES analysis identified forty-three mutations that had not been reported previously, one of which was present in two of the four tumors. WES analysis also corroborated twenty-two mutations that were reported previously. No single mutation occurred in all four tumors or in all stage 4 tumors. Three of the four tumors harbored genes with CADD scores ≥20, indicative of mutations associated with human pathologies. The average depth of coverage ranged from 39.68 to 90.27, with >99% sequences mapping to the genome. In summary, WES identified sixty-five coding mutations including forty-three mutations not reported previously in primary neuroblastoma tumors. The three stage 4 tumors contained mutations in genes encoding protein products that regulate immune function or cell adhesion and tumor cell metastasis.
Abstract The hydrodynamics of gas‐solid flow, usually referred to as circulating fluidizedbed flow, was studied in a 7.5‐cm clear acrylic riser with 75‐μm FCC catalyst particles. Data were obtained for three central sections as a function of gas and solids flow rates. Fluxes were measured by means of an extraction probe. Particle concentrations were measured with an X‐ray densitometer. In agreement with previous investigators, these data showed the flow to be in the core‐annular regime, with a dilute rising core and a dense descending annular region. However, unlike the previous studies conducted worldwide, the data obtained in this investigation allowed us to determine the viscosity of the suspension. The viscosity was a linear function of the volume fraction of solids. It extrapolates to the high bubbling‐bed viscosities.
We reported previously that the BET inhibitor (BETi) JQ1 decreases levels of the DNA repair protein RAD51 and that this decrease is concomitant with increased levels of DNA damage. Based on these findings, we hypothesized that a BETi would augment DNA damage produced by radiation and function as a radiosensitizer. We used clonogenic assays to evaluate the effect of JQ1 ± ionizing radiation (IR) on three pancreatic cancer cell lines in vitro. We performed immunofluorescence assays to assess the impact of JQ1 ± IR on DNA damage as reflected by levels of the DNA damage marker γH2AX, and immunoblots to assess levels of the DNA repair protein RAD51. We also compared the effect of these agents on the clonogenic potential of transfectants that expressed contrasting levels of the principle molecular targets of JQ1 (BRD2, BRD4) to determine whether levels of these BET proteins affected sensitivity to JQ1 ± IR. The data show that JQ1 + IR decreased the clonogenic potential of pancreatic cancer cells more than either modality alone. This anticlonogenic effect was associated with increased DNA damage and decreased levels of RAD51. Further, lower levels of BRD2 or BRD4 increased sensitivity to JQ1 and JQ1 + IR, suggesting that pre-treatment levels of BRD2 or BRD4 may predict sensitivity to a BETi or to a BETi + IR. We suggest that a BETi + IR merits evaluation as therapy prior to surgery for pancreatic cancer patients with borderline resectable disease.
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.
Abstract Cholangiocarcinoma (CCA) is a lethal malignancy arising from cholangiocytes in any part of the biliary tree. The incidence of CCA has been on the rise worldwide, and the prognosis and clinical outcome have remained essentially unchanged for 30 years. The majority of patients are diagnosed at late stage, and surgery continues to be the only cure. Patients receive systemic chemotherapy with the first-line combination therapy comprising gemcitabine and cisplatin. Median survival for these patients is <12 months, emphasizing the need to improve current treatment. A step toward improving outcome is the pre-clinical evaluation of novel therapeutics. Recently the bromodomain and extra-terminal domain (BET) inhibitor JQ1 has been shown to suppress tumor growth in preclinical models of multiple tumor types. The therapeutic effect of JQ1 has been attributed, at least in part, to its ability to inhibit c-Myc expression. 95% of CCA tumors express c-Myc and its down-regulation has been shown to reduce the invasive potential of CCA cells, suggesting that c-Myc contributes to CCA phenotype. Our lab recently evaluated the efficacy of the BET inhibitor JQ1 in CCA patient-derived xenograft mouse models. JQ1 suppressed CCA tumor growth in 2 of the 3 models. To determine gene products whose upregulation or downregulation is responsible for the differences in sensitivity to JQ1 among our CCA models, we generated expression profiles of tumors from vehicle control and JQ1 treated mice using NanoString technology (nCounter PanCancer Pathways panel). Our data demonstrate that JQ1 inhibited the expression of c-Myc to a greater extent in the sensitive models than in the insensitive model. Expression array data showed further that gene products involved in cell cycle and DNA repair pathways were also decreased by JQ1. Of particular interest were two transcriptional targets of c-Myc, Chk1 and BRCA2, each of which is involved in DNA damage response. Immunohistochemistry staining confirmed expression profile analyses. We conclude that the inhibition of cell cycle and DNA repair genes may contribute to the mechanism of action of JQ1 in CCA tumors. Citation Format: Aubrey L. Miller, Patrick L. Garcia, Tracy L. Gamblin, Leona N. Council, Xiangqin Cui, James E. Bradner, Eddy S. Yang, Karina J. Yoon. JQ1 sensitivity of patient-derived xenograft models of cholangiocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1293.
Abstract Cholangiocarcinoma (CCA) is an aggressive bile duct neoplasm, and the second most common primary hepatic malignancy. CCA is usually diagnosed at a late stage where the current standard of care, resection followed by gemcitabine with or without cisplatin, is not effective. Up to 90% of CCA patients are ineligible for resection and, of those eligible, postoperative chemotherapy does not significantly prolong overall survival. The limited number of CCA models hinders drug development and discovery of new therapeutic targets. Mutations have been identified in KRAS (17% of CCA tumors), p53 (44%) and SMAD4 (17%), but are not recognized as essential to CCA progression. c-Myc protein is highly expressed in 94% of CCA cases while low to undetectable in normal adult liver, suggesting c-Myc overexpression may contribute to CCA tumor progression but this hypothesis has not been tested. Toward developing novel effective therapies for CCA, we evaluated the effect of the bromodomain and extraterminal domain (BET) inhibitor JQ1 on c-Myc expression and on the proliferation of CCA cells. JQ1 inhibits c-Myc transcription by binding to the acetylated lysine (K-Ac) binding pockets of BET protein family members (BRD2, BRD3, BRD4 and BRDT), preventing their interaction with K-Ac residues on nuclear proteins, disrupting the formation of functional transcriptional complexes and inhibiting c-Myc transcription. We have shown that JQ1 inhibited tumor growth in a patient-derived xenograft (PDX) model of CCA and downregulated expression of c-Myc and its transcriptional targets BRCA2 and Chk1, both involved in DNA damage response. Our current data show that JQ1 decreased c-Myc protein expression, CCA cell viability, and clonogenic potential in KKU-055 CCA cell line. Of note, the literature suggests that tumors deficient in BRCA2 or Chk1 are relatively sensitive to PARP inhibitors (PARPi). Therefore, we hypothesize that JQ1-induced downregulation of c-Myc and its transcriptional targets BRCA2 and Chk1 would potentiate the efficacy of PARPi. We exposed CCA cell line KKU-055 with a range of JQ1 and PARPi (olaparib and veliparib) concentrations as single agent or combination therapy. Each combination of JQ1 with either PARPi was more effective than any of the three drugs as single agents. Combination indices (CI), calculated using CompuSyn 1.0, ranged from 0.05 to 0.09 which indicated strong synergy for JQ1 with olaparib or veliparib. Further, synergy with JQ1 was not dependent on a single PARPi. JQ1 with olaparib (1:1) also decreased the clonogenic potential of KKU-055 cells, compared to each drug as a single agent. These data suggest that inhibition of BET protein function sensitizes CCA cells to PARPi, coincident with downregulation of c-Myc and its transcriptional targets BRCA2 and Chk1. Research funded by NIH/NCI R21 CA205501 Citation Format: Samuel C. Fehling, Aubrey L. Miller, James E. Bradner, Karina J. Yoon. The BET inhibitor JQ1 sensitizes cholangiocarcinoma cells to PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5824.
