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Our previous finding that the BET inhibitor (BETi) JQ1 increases levels of the DNA damage marker γH2AX suggested that JQ1 might enhance the sensitivity of tumor cells to PARP inhibitors (PARPi), which are selectively toxic to cells that harbor relatively high levels of DNA damage. To address this hypothesis, we evaluated the effect of a BETi (JQ1 or I-BET762) combined with a PARPi (olaparib or veliparib) in KKU-055 and KKU-100 cholangiocarcinoma (CCA) cell lines and of JQ1 with olaparib in a xenograft model of CCA. Each combination was more effective than any of the four drugs as single agents. Combination indices ranged from 0.1 to 0.8 at the ED50 for all combinations, indicating synergy and demonstrating that synergy was not limited to a specific combination. Mechanistically, downregulation of BETi molecular targets BRD2 or BRD4 by shRNA sensitized CCA cells to BETi as single agents as well as to the combination of a BETi + a PARPi. Our data indicate that combinations of a BETi with a PARPi merit further evaluation as a promising strategy for CCA.

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 &lt, 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.

Ovarian cancer is the fifth leading cause of cancer-related deaths among women in the United States. Although most patients respond to frontline therapy, virtually all patients relapse with chemoresistant disease. This study addresses the hypothesis that carboplatin or tamoxifen + FTY720, a sphingosine analogue, will minimize or circumvent drug-resistance in ovarian cancer cells and tumor models. In vitro data demonstrate that FTY720 sensitized two drug-resistant (A2780. cp20, HeyA8. MDR) and two high-grade serous ovarian cancer cell lines (COV362, CAOV3) to carboplatin, a standard of care for patients with ovarian cancer, and to the selective estrogen receptor modulator tamoxifen. FTY720 + tamoxifen was synergistic in vitro, and combinations of FTY720 + carboplatin or + tamoxifen were more effective than each single agent in a patient-derived xenograft model of ovarian carcinoma. FTY720 + tamoxifen arrested tumor growth. FTY720 + carboplatin induced tumor regressions, with tumor volumes reduced by ∼86% compared to initial tumor volumes. Anti-tumor efficacy was concomitant with increases in intracellular proapoptotic lipid ceramide. The data suggest that FTY720 + tamoxifen or carboplatin may be effective in treating ovarian tumors.

Previously, we determined microRNA-31 (miR-31) is a non-coding tumor suppressive gene frequently deleted in glioblastoma (GBM); miR-31 suppresses tumor growth, in part, by limiting the activity of NF-κB. Herein, we expand our previous studies by characterizing the role of miR-31 during neural precursor cell (NPC) to astrocyte differentiation. We demonstrate that miR-31 expression and activity is suppressed in NPCs by stem cell factors such as Lin28, c-Myc, SOX2 and Oct4. However, during astrocytogenesis, miR-31 is induced by STAT3 and SMAD1/5/8, which mediate astrocyte differentiation. We determined miR-31 is required for terminal astrocyte differentiation, and that the loss of miR-31 impairs this process and/or prevents astrocyte maturation. We demonstrate that miR-31 promotes astrocyte development, in part, by reducing the levels of Lin28, a stem cell factor implicated in NPC renewal. These data suggest that miR-31 deletions may disrupt astrocyte development and/or homeostasis.

In glioma, microglia and macrophages are the largest population of tumor-infiltrating cells, referred to as glioma associated macrophages (GAMs). Herein, we sought to determine the role of Suppressor of Cytokine Signaling 3 (SOCS3), a negative regulator of Signal Transducer and Activator of Transcription 3 (STAT3), in GAM functionality in glioma. We utilized a conditional model in which SOCS3 deletion is restricted to the myeloid cell population. We found that SOCS3-deficient bone marrow-derived macrophages display enhanced and prolonged expression of pro-inflammatory M1 cytokines when exposed to glioma tumor cell conditioned medium in vitro. Moreover, we found that deletion of SOCS3 in the myeloid cell population delays intracranial tumor growth and increases survival of mice bearing orthotopic glioma tumors in vivo. Although intracranial tumors from mice with SOCS3-deficient myeloid cells appear histologically similar to control mice, we observed that loss of SOCS3 in myeloid cells results in decreased M2 polarized macrophage infiltration in the tumors. Furthermore, loss of SOCS3 in myeloid cells results in increased CD8+ T-cell and decreased regulatory T-cell infiltration in the tumors. These findings demonstrate a beneficial effect of M1 polarized macrophages on suppressing glioma tumor growth, and highlight the importance of immune cells in the tumor microenvironment.

Cytochrome P450 enzymes are hemeproteins that catalyze the monooxygenation of a wide-range of structurally diverse substrates of endogenous and exogenous origin. These heme monooxygenases receive electrons from NADH/NADPH via electron transfer proteins. The cytochrome P450 enzymes, which constitute a diverse superfamily of more than 8,700 proteins, share a common tertiary fold but 55% and Bhattacharyya coefficient > 80%), despite the low sequence identity (< 25%) and sequence similarity (< 50%) across the cytochrome P450 superfamily. Noticeable differences in Cα atom fluctuations of structural elements responsible for substrate binding were noticed. These differences in residue fluctuations might be crucial for substrate selectivity in these enzymes.

Inter-domain communication plays a key role in the function of modular proteins. Earlier studies have demonstrated that the coupling of domain motions is important in mediating site-to-site communications in modular proteins. In the present study, bioinformatics and molecular simulations were usedto trace “pre-existing” residue-residue interaction networks that mediate coupled-domain dynamics in multi-domain Escherichia coli methionyl-tRNA synthetase (Ec MetRS). In particular, a comparative study was carried out to evaluate the effectiveness of coarse-grained normal mode analysis and all-atom molecular dynamic simulation in predicting pre-existing pathways of inter-domain communications in this enzyme. Integration of dynamic information of residues with their evolutionary features (conserved and coevolved) demonstrated that multiple residue-residue interaction networks exist in Ec MetRS that promote dynamic coupling between the anticodon binding domain and the connective polypeptide I domain, which are > 50Å apart, through correlated motions. Mutation of residues on these pathways have distinct impact on the dynamics and function of this enzyme. Moreover, the present study revealed that the dynamic information obtained from the coarse-grained normal mode analysis is comparable to the atomistic molecular dynamics simulations in predicting the interaction networks that are essential for promoting coupled-domain dynamics in Ec MetRS. KEYWORDS: Domain-domain Communication; Molecular Dynamics; Methionyl-tRNA Synthetase; Normal Mode Analysis; Coupled-domain Dynamics; Course-grained Normal Mode Analysis; Aminoacyl tRNA Synthetases; Statistical Coupling Analysis

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.

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 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.

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.

Like many cancers, glioma cells have an altered metabolic state and utilize glycolysis even in the presence of oxygen, and thus require a large amount of glucose. The ketogenic diet is a high fat, low carbohydrate, low protein diet that limits the amount of glucose, and theoretically starves tumor growth. In addition, recent reports have indicated that the gut microbiome can influence tumor growth as well as the effectiveness of certain therapeutic agents. In this study, we sought to determine if the ketogenic diet is an effective treatment for glioma, and to correlate changes in the healthy gut bacteria in response to the ketogenic diet. In an orthotopic mouse model of glioma, we found that mice fed the ketogenic diet had slightly increased survival compared to mice fed a normal diet. Comparing the composition of gut bacteria from animals on normal versus ketogenic diets, we found significant differences in several key microbes. Interestingly, we observed that there were several long-term ketogenic survivors implanted with intracranial tumors and displayed a significant increase in the percentage of microbes found in the murine gastrointestinal tract (F. rodentium and related microbes). We found that F. rodentium growth does not occur until day 21 on the diet; thus, F. rodentium could be introduced early as a probiotic and enhance anti-tumor effects of the ketogenic diet. Additionally, we found that the ketogenic diet increased the percentage of mature Th17 cells, potentially collaborating with the growth of F. rodentium to decrease tumor growth. No studies have ever evaluated a probiotic for therapeutic efficacy in glioma. As the ketogenic diet is very strict, and can negatively affect quality of life, a ketogenic-diet probiotic (healthy gut bacteria, similar to the probiotics in yogurt) may offer an alternative supplement and patients could possibly eat a modified (less strict) ketogenic diet.

Coarse-grained simulations have emerged as invaluable tools for studying conformational changes in biomolecules. To evaluate the effectiveness of computationally inexpensive coarse-grained models in studying global and local dynamics of large protein systems like aminoacyl-tRNA synthetases, we have performed coarse-grained normal mode analysis, as well as principle component analysis on trajectories of all-atom and coarse-grained molecular dynamics simulations for three aminoacyl-tRNA synthetases—Escherichia coli methionyl-tRNA synthetase, Thermus thermophilus leucyl-tRNA synthetase, and Enterococcus faecium prolyl-tRNA synthetase. In the present study, comparison of predicted dynamics based on B-factor and overlap calculations revealed that coarse-grained methods are comparable to the all-atom simulations in depicting the intrinsic global dynamics of the three enzymes. However, the principal component analyses of the motions obtained from the all-atom molecular dynamics simulations provide a superior description of the local fluctuations of these enzymes. In particular, the all-atom model was able to capture the functionally relevant substrate-induced dynamical changes in prolyl-tRNA synthetase. The alteration in the coupled dynamics between the catalytically important proline-binding loop and its neighboring structural elements due to substrate binding has been characterized and reported for the first time. Taken together, the study portrays comparable and contrasting situations in studying the functional dynamics of large multi-domain aminoacyl-tRNA synthetases using coarse-grained and all-atom simulation methods. Figure Substrate-induced conformational change in E. facium prolyl-tRNA synthetase Electronic supplementary material The online version of this article (doi:10.1007/s00894-014-2245-1) contains supplementary material, which is available to authorized users.

Glioblastoma (GBM), a primary Grade IV astrocytoma, is the most aggressive primary brain cancer in humans. Despite combined radiotherapy and chemotherapy, the median survival is 14 months. We and others have validated that levels of phosphorylated JAK1/2 and STAT-3 are increased in GBM tissues. When stimulated with IL-6 family members, JAK1/2 recruits and phosphorylates STAT-3. Phosphorylated STAT-3 dimerizes and translocates to the nucleus where it regulates genes involved in proliferation, angiogenesis, anti-apoptotic activity and immunosuppression. Elevated JAK/STAT activation is generated through an increase in IL-6 family cytokines as well as a decrease in JAK/STAT-3 negative regulators. To therapeutically inhibit the JAK/STAT-3 pathway in GBM we employed ruxolitinib, an FDA approved JAK1/JAK2 inhibitor. Human GBM cell lines U87-MG and U251-MG, as well as patient derived GBM xenograft lines JX6 and JX12, were treated with ruxolitinib in doses ranging from 0.01 to 10 μM. Ruxolitinib was observed to inhibit STAT-3 activation in a dose-dependent manner. Pre-treatment of U87-MG and U251-MG cells with ruxolitinib inhibited stimulus-induced STAT-3 activation and downstream gene expression in a dose-dependent manner. Future studies will examine ruxolitinib effects on migration, invasion, apoptosis and proliferation in vitro. Collectively, these data indicate the potential for ruxolitinib therapy for GBM patients. Citation Format: Samuel Fehling, Braden McFarland, Etty Benveniste. Ruxolitinib inhibits STAT-3 activation in glioblastoma. [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 3861.

Glioblastomas (GBMs) are deadly tumors of the central nervous system. Most GBM exhibit homozygous deletions of the CDKN2A and CDKN2B tumor suppressors at 9p21.3, although loss of CDKN2A/B alone is insufficient to drive gliomagenesis. MIR31HG, which encodes microRNA-31 (miR-31), is a novel non-coding tumor suppressor positioned adjacent to CDKN2A/B at 9p21.3. We have determined that miR-31 expression is compromised in >72% of all GBM, and for patients, this predicts significantly shortened survival times independent of CDKN2A/B status. We show that miR-31 inhibits NF-κB signaling by targeting TRADD, its upstream activator. Moreover, upon reintroduction, miR-31 significantly reduces tumor burden and lengthens survival times in animal models. As such, our work identifies loss of miR-31 as a novel non-coding tumor-driving event in GBM.