bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C28: The combination of Acetazolamide and Sulforophane targets the tumor initiating cells in human bronchial carcinoids
Authors: Baluch Narges , Bayat Mokhtari Reza , Homayouni Tina , Akbari Pedram , Heshmati Fatemeh , Islam Syed S , Kumar Sushil , Aitken Karen , Fitzpatrick Carrie , Yeger Herman
Published: May 2016

Bronchial carcinoids (BC) are neuroendocrine tumors that present as typical (TC) and atypical (AC) variants, the latter being more aggressive. The AC form of BC possesses a higher degree of invasiveness and metastatic potential. It is important to understand how the BC tumor initiating cells (TIC) or cancer stem cells (CSC) in BC account for this phenotype. In order to functionally characterize the suspected BC stem cell fraction, BC cell lines (H727 and H720) were grown under a non-adhesive condition to form 3D spheroids favouring expansion of the TIC. The third generation of spheroids was assessed for clonogenicity, tumorigenicity (xenograft model), stem cell and hypoxia markers, drug resistance, karyotype and microarray analysis compared to parental cells. We previously showed that a pan-carbonic anhydrase inhibitor, acetazolamide (AZ) significantly potentiated the anti-tumor effects of sulforophane (SFN, a natural isothiocyanate with HDACi activity) on these lung carcinoid cell lines in vitro and subcutaneous xenografts. Here we developed an orthotopic lung model to study tumor progression and metastatic behaviour of the spheroid forming BC cells. Orthotopically grown tumors were treated with AZ, SFN and the AZ+SFN combination using previous doses within clinically available ranges. Results demonstrated that the spheroid grown BC cells showed increased clonogenic, tumorigenic, and drug resistance potential, with enhanced expression of stem cell, carbonic anhydrase and hypoxia markers as compared with the control non-spheroid cells, all suggesting selection for the TIC or CSC fraction. As in the subcutaneous model the combination of AZ with SFN was more effective than either single agent alone in inhibiting orthotopic tumor growth and survival. Taken together the evidence indicates that the novel AZ+SFN combination is likely targeting the TIC/CSC population. This offers increased value in terms of the management of BC tumor progression and possible metastasis in BC patients presenting with more aggressive variants.

Citation Format: Narges Baluch, Reza Bayat Mokhtari, Tina Homayouni, Pedram Akbari, Fatemeh Heshmati, Syed S Islam, Sushil Kumar, Karen Aitken, Carrie Fitzpatrick, Herman Yeger. The combination of Acetazolamide and Sulforophane targets the tumor initiating cells in human bronchial carcinoids. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C28.
Abstract C39: First-in-class inhibitors of the putatively undruggable DNA repair target Poly(ADP-ribose) glycohydrolase (PARG)
Authors: Waszkowycz Bohdan , James Dominic , Acton Ben , Fairweather Emma , Fritzl Sam , Hamilton Niall , Hamilton Nicola , Holt Sarah , Hitchen James , Hutton Colin , Jones Stuart , Jordan Allan , McGonagle Alison , Mould Daniel , Small Helen , Smith Kate , Stowell Alexandra , Waddell Ian D. , Ogilvie Donald
Published: May 2016

Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose) polymerases (PARPs). PARG depletion, using RNAi, results in several effects such as PAR chain persistence, progression of single- to double-strand DNA lesions and NAD+ depletion. Given these findings, inhibition of PARG with a small molecule agent offers a potential opportunity to interfere with DNA repair mechanisms and induce cell death in those cells with increased susceptibility to DNA damage, such as tumour cells.

Previous efforts to develop small molecule inhibitors of PARG activity have generally been hampered by poor physicochemical properties, off-target pharmacology and a lack of cell permeability, leading some to suggest that PARG may be undruggable.

In contrast, we have now developed a series of first-in-class PARG inhibitors which display drug-like properties and attractive pharmacokinetic parameters. These compounds have proved to be useful biological tool compounds.

Moreover, displaying selective activity in both biochemical and, more importantly, cellular assays of PARG function, these derivatives have allowed an exploration of the phenotypes resulting from reversible, pharmacological PARG inhibition in both in vitro cell panels and in vivo models. Furthermore, our initial bioinformatic analysis suggests that deficiency of a known tumour suppressor confers sensitivity to PARG inhibition, suggesting patient populations that will potentially benefit from PARGi therapies.

Citation Format: Bohdan Waszkowycz, Dominic James, Ben Acton, Emma Fairweather, Sam Fritzl, Niall Hamilton, Nicola Hamilton, Sarah Holt, James Hitchen, Colin Hutton, Stuart Jones, Allan Jordan, Alison McGonagle, Daniel Mould, Helen Small, Kate Smith, Alexandra Stowell, Ian D. Waddell, Donald Ogilvie. First-in-class inhibitors of the putatively undruggable DNA repair target Poly(ADP-ribose) glycohydrolase (PARG). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C39.
Abstract C60: Pre-clinical efficacy of the ATR inhibitor AZD6738 in combination with the PARP inhibitor olaparib
Authors: Lau Alan , Brown Elaine , Thomason Andrew , Odedra Rajesh , Sheridan Victoria , Cadogan Elaine , Xu Shirlian , Cui Andy , Gavine Paul R. , O'Connor Mark
Published: May 2016

The PARP inhibitor olaparib acts through both inhibition of DNA single-strand-break repair and trapping of PARP-DNA complexes creating DNA lesions which cause replication fork stalling and collapsed fork DNA breaks. Cells which have lost BRCA-dependent homologous recombination repair are highly sensitive to olaparib treatment and this has led to its approval in patients with tumors carrying BRCA mutations. In addition, ATM (Ataxia telangiectasia mutated) and ATR (Ataxia telangiectasia mutated and Rad3 related) dependent DNA repair processes are hypothesized to be important survival pathways to PARP inhibitor treatment. In pre-clinical studies cancer cells which have defects in either ATM or ATR have been shown to be sensitive to PARP inhibitors. Here we present data that the orally bioavailable ATR inhibitor AZD6738 (in Phase-I clinial trials) combines synergistically with olaparib leading to cell death and anti-tumour activity in pre-clinical models. The combinations are effective across a panel of gastric and lung cancer cell lines in vitro. In addition results from isogenic ATM−/-/− knockout versus ATM+/+/+FaDu head and neck cancer cell line pairs show enhanced combination activity in ATM knockout cells versus ATM wild-type cells. Studies in vivo show that through intermittent dosing the combination is tolerated while demonstrating significant anti-tumour efficacy and regressions across multiple human patient derived primary explant models. Together, these data support the notion of development of AZD6738 and olaparib combinations for the treatment of ATM-deficient cancers.

Citation Format: Alan Lau, Elaine Brown, Andrew Thomason, Rajesh Odedra, Victoria Sheridan, Elaine Cadogan, Shirlian Xu, Andy Cui, Paul R. Gavine, Mark O'Connor. Pre-clinical efficacy of the ATR inhibitor AZD6738 in combination with the PARP inhibitor olaparib. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C60.
Abstract C85: Identification of a novel potent selective SMYD3 inhibitor with oral bioavailability
Authors: Mitchell Lorna H. , Boriack-Sjodin Paula A. , Smith Sherri , Thomenius Michael , Rioux Nathalie , Munchhof Michael , Mills James E. , Klaus Christine , Totman Jennifer , Riera Thomas V. , Raimondi Alejandra , Jacques Suzanne L. , Foley Megan , Waters Nigel J. , Kuntz Kevin W. , Wigle Tim J. , Porter Scott Margaret , Copeland Robert A. , Smith Jesse J. , Chesworth Richard
Published: May 2016

SMYD3 (Set and Mynd Domain containing 3) is a lysine methyltransferase overexpressed in several cancer types including breast, prostrate, pancreatic, and lung, and this overexpression is associated with poor clinical prognosis. Genetic knockdown of SMYD3 by shRNA has been shown to decrease proliferation in a range of cancer cell lines suggesting that inhibition of SMYD3 may have therapeutic utility.

In this presentation we describe the discovery and optimization of a novel series of oxindole sulfonamides and sulfamides with SMYD3 inhibitory activity. One of these compounds, EPZ030456, has a SMYD3 biochemical IC50 of 4 nM and is active in cells with an IC50 of 48 nM in a trimethyl MAP3K2 (MEKK2) in-cell western (ICW) assay. The crystal structure of this compound was solved with SMYD3 and the nucleotide substrate, S-adenosylmethionine and shows the oxindole portion of the molecule extends into the SMYD3 lysine binding channel. EPZ030456 shows < 30% inhibition at a 10 uM screening concentration against 17 histone methyltransferase targets tested, including SMYD2.

Further optimization within the series resulted in EPZ031686 which has similar potency to EPZ030456 with a biochemical IC50 of 3 nM and an ICW IC50 of 36 nM and in addition exhibits good bioavailability following oral dosing in mice. Hence, EPZ031686 is a suitable tool to study the role of SMYD3 in cancer and other therapeutic areas, using both in vitro and in vivo models.

Citation Format: Lorna H. Mitchell, Paula A. Boriack-Sjodin, Sherri Smith, Michael Thomenius, Nathalie Rioux, Michael Munchhof, James E. Mills, Christine Klaus, Jennifer Totman, Thomas V. Riera, Alejandra Raimondi, Suzanne L. Jacques, Megan Foley, Nigel J. Waters, Kevin W. Kuntz, Tim J. Wigle, Margaret Porter Scott, Robert A. Copeland, Jesse J. Smith, Richard Chesworth. Identification of a novel potent selective SMYD3 inhibitor with oral bioavailability. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C85.
Abstract C88: HPV infections, hTERT methylation and hTERT expression in patients with invasive cervical cancer
Authors: Moreno-Acosta Pablo , Morales Nicolas , Burgos Marcela , Gamboa Oscar , Mejia Juan Carlos , Romero-Rojas Alfredo , Combita Alba Lucia , Molano Monica
Published: May 2016

Background: There is limited information of the role of hTERT methylation, hTERT expression and their association with type specific HPV infections in patients with invasive cervical cancer

Objective: To analyze the possible association between methylation status of the hTERT promoter gene, hTERT expression and HPV infection in patients with invasive cervical cancer.

Methodology: Eighty seven frozen samples of women with invasive cervical cancer were analyzed for type specific HPV infection using a GP5+/GP6+ mediated PCR- RLB. hTERT DNA methylation analysis was performed on bisulfite modified DNA using a new PCR-RLB-hTERT methylation assay targeting two regions flanking the hTERT [region 1 (nt -240 to -1) and region 2 (nt +1 to +120) relative to first ATG]. Expression of hTERT was detected by immunohistochemistry using an Anti-hTERT (348-358) rabbit pAb).

Results: All samples with HPV types belonging to the α7 species (HPV 18, 45 and 59) showed no hTERT methylation in region 1 (core promoter) and an increase in% of partial methylation in region 2. Samples with HPV types belonging to the α9 specie (16, 52, 35, 31 and 58) showed similar% of methylation in region 1 and region 2 according viral type but the percentages of methylation were different between them (ranging from 0% for HPV58 samples to 66.7% for HPV31). Strong expression of hTERT was observed in 77% of the samples independent of the HPV genotype.

Conclusion: hTERT methylation seems to be associated with specific genotype HPV infection; however expression of the hTERT protein seems to depend of additional molecular events and worth further investigation.

Citation Format: Pablo Moreno-Acosta, Nicolas Morales, Marcela Burgos, Oscar Gamboa, Juan Carlos Mejia, Alfredo Romero-Rojas, Alba Lucia Combita, Monica Molano. HPV infections, hTERT methylation and hTERT expression in patients with invasive cervical cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C88.
Abstract CN02-03: Heterogeneity, drug resistance, and clonal dynamics in colorectal cancers
Authors: Bardelli Alberto
Published: May 2016

Colorectal cancers evolve by a reiterative process of genetic diversification and clonal evolution. Tissue and liquid biopsies can be used to define CRC molecular subtypes and to monitor clonal evolution during therapy. Using these approaches, CRC patients were found to respond selectively to targeted agents interfering with oncogenic nodes of the EGFR signaling pathway. Notably, the patient-specific responses can be recapitulated and paralleled in cellular and mouse clinical proxies (CRC-avatars). The inevitable development of acquired resistance to inhibitors of the EGFR signaling axis presently limits further clinical advances. Strategies to prevent or overcome resistance are therefore essential to design the next generation of molecularly-driven clinical trials for CRC patients.

Citation Format: Alberto Bardelli. Heterogeneity, drug resistance, and clonal dynamics in colorectal cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr CN02-03.
Abstract LB-B16: In vitro monitoring of the efficacy of cancer immunotherapy designs trough an impedance-based platform
Authors: Cerignoli Fabio , Zhu Jenny , Xi Biao , Zhao Leyna , Irelan Jeff , Abassi Yama
Published: May 2016

Immunotherapy is emerging as one of the most promising approaches in cancer treatment, allowing specific targeting of cancer cells from CD8+ T Lymphocytes based on surface marker expression. In vitro characterization of the efficacy and potency of BiTEs or CAR T-Cell constructs is a necessary step before moving to animal models and Phase I and II clinical studies. In this study we present the optimization of several protocols to analyze cancer immunotherapy reagents using an impedance-based platform. The platform allows detection of cell adhesion and cell death trough monitoring the change in conductance of microelectrodes embedded on the bottom of the culture dish. Because cell adhesion over the electrodes alters the conductivity of the surface, any change in cell morphology, adhesion or number is detected as alteration of an impedance-related parameter called Cell index (Ci). Furthermore, because the platform avoid the use of any chemical label or sample processing, it allows continuous monitoring of cell activity. Dose-responses curves of BiTEs antibodies against epithelial tumor cell lines with a range of expression of the EpCAM antigen show correlation between surface expression level and efficacy of the constructs. Decreasing ratios between effector and target cells, in combination with longer time of monitoring reveal the efficacy of such constructs also at equivalent stoichiometric ratio. Taken together our results demonstrate higher sensitivity of the impedance-based approach over established methodologies like Chromium-51 release assay and live-cell imaging, improving and speeding up the workflow for therapy optimization.

Citation Format: Fabio Cerignoli, Jenny Zhu, Biao Xi, Leyna Zhao, Jeff Irelan, Yama Abassi. In vitro monitoring of the efficacy of cancer immunotherapy designs trough an impedance-based platform. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B16.
Abstract LB-C20: Lead generation and optimisation of a series of novel glutaminase (GLS) inhibitors
Authors: Charles Mark D. , Cairnduff Ceri , Brookfield Joanna , Sabin Verity , Ekwuru Tennyson , Jones Neil , Ting Attilla , Smith James , Nissink Willem , Finlay Ray , Powell Steve , Critchlow Susan , Ward Richard , Wood Matthew , Ruston Linette , Winter Jon , Hollingsworth Ian
Published: May 2016

Glutamine is an essential nutrient for cancer cells and is used to support cancer cell growth. Glutamine is converted to glutamate by the enzyme glutaminase, which is then converted into alpha-ketoglutarate, a metabolic intermediate within the TCA cycle which serves as a precursor for the biosynthesis of ATP, NADPH, fatty acids, glutathione, nucleic acids and amino acids. As such, we regarded glutaminolysis as a key pathway for therapeutic intervention.

At the outset of the project there were several reported inhibitors of glutaminase known in the literature; however, they possess undesirable drug like properties. We describe here our efforts to discover novel inhibitors of glutaminase with a promising development profile. From a high throughput screen of approximately 900,000 compounds in the AstraZeneca collection, we were able to identify three series of glutaminase inhibitors. One of these series has been optimised to deliver cell potent, selective and orally bioavailable inhibitors of glutaminase 1.

Citation Format: Mark D. Charles, Ceri Cairnduff, Joanna Brookfield, Verity Sabin, Tennyson Ekwuru, Neil Jones, Attilla Ting, James Smith, Willem Nissink, Ray Finlay, Steve Powell, Susan Critchlow, Richard Ward, Matthew Wood, Linette Ruston, Jon Winter, Ian Hollingsworth. Lead generation and optimisation of a series of novel glutaminase (GLS) inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C20.
Abstract PL03-03: K-RAS dependency in pancreatic cancer
Authors: Tuveson David A.
Published: May 2016

2015 AACR-NCI-EORTC

K-RAS dependency in Pancreatic Cancer

David Tuveson, M.D., Ph.D., Cold Spring Harbor Laboratory

Efforts to inhibit the KRAS oncogene in pancreatic cancer have focused on classical downstream pathways and empirically identified dependencies. We have developed organoid models of mouse and human pancreatic cancer to expedite such translational approaches for this disease. We find that organoid models of PDA demonstrate ERBB pathway activation in response to dual MEK/AKT inhibition, and that this feature is absent in non-neoplastic organoids. Furthermore, organoid models predict the limited activity of EGFR specific inhibitors in PDA patients that are also receiving MEK or AKT inhibitors. These models have also been an invaluable resource for probing new dependencies in PDA, and will be discussed.

Citation Format: David A. Tuveson. K-RAS dependency in pancreatic cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL03-03.
Abstract PL07-02: Tumor hypoxia and radiation resistance: Anything new on the horizon?
Authors: Deutsch Eric , Chargari CYrus
Published: May 2016

Hypoxia is one of the key factors influencing clinical outcome after radiotherapy responsible for reduced local control that will influence overall survival, as may the hypoxic conditions by increasing malignant progression. In sharp contrast with the progresses and the rapid diffusion of functional imaging to characterize hypoxic tumors and the fact that experimental correction of tumor tumor hypoxia reverses tumor resistance to irradiation, there is no approved concomitant therapy in order to maximize response to radiotherapy for the treatment of hypoxic tumors. For decades, hypoxia was thought to act primarily on intrinsic tumor cells resistance, namely the number of clonogenic cancer stem cells surviving after radiation treatment. In the meantime, improvements into the understanding of the mechanisms triggered by hypoxia have unveiled novel molecular targets for potential combination to radiation therapy such as the interplay between STAT3 signaling and adaptation to hypoxia while the impact of tumor hypoxia on tumor microenvironment changes have expand to the immune network. Increased cellular turnover and hypoxia promote the production and release of large amounts of immunosupressive adenosine into the local microenvironment. Hypoxia can induce HIF-1a-dependent expression of arginase-1 and M2 polarization of macrophages. The spectrum of candidate therapies to target hypoxic tumors has moved from bioreductive cytotoxic agents to HIF-1, STAT3 inhibitors while growing number of immunological tools are becoming available for targeting the immune component. The ongoing EORTC 1219-ROG-HNCG/DAHANCA project 29 has gained form the past clinical attempts to target hypoxic tumors and will provide the methodology way for future clinical development of such novel combos based on optimal patients selection using biological and/or imaging criteria.

Citation Format: Eric Deutsch, CYrus Chargari. Tumor hypoxia and radiation resistance: Anything new on the horizon? [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL07-02.
Abstract PR08: ARV-330: An androgen receptor PROTAC degrader for prostate cancer
Authors: Winkler James D. , Jin Meizhong , Crew Andy P. , Rossi AnnMarie K. , Willard Ryan R. , Dong Hanqing , Siu Kam , Wang Jing , Gordon Deborah A. , Chen Xin , Ferraro Caterina , Crews Craig M. , Coleman Kevin , Neklesa Taavi K.
Published: May 2016

Patients with prostate cancer who progress on therapy often have enhanced Androgen Receptor (AR) signaling due to several mechanisms: increased androgen production, increased AR expression, and/or specific AR mutations that render current therapies ineffective. A novel approach to block AR signaling is to specifically target AR for degradation. To do this, we have created AR PROTACs (PROtein-TArgeting Chimeras), bi-functional molecules that have an AR binding moiety on one end and an E3 ligase-recruiting element on the other end, which leads to AR ubiquitination and degradation. We have applied this technology to determine whether it could address mechanisms of resistance to current therapy in prostate cancer models.

Our lead AR PROTAC, ARV-330, degrades AR in LNCaP and VCaP cells with 50% degradation concentrations (DC50s) < 1nM. AR degradation had functional consequences in cells, suppressing the AR target gene PSA, inhibiting proliferation, and potently inducing apoptosis in VCaP cells, with maximal apoptosis observed around 20 nM, versus 1 uM for enzalutamide. While both ARV-330 and enzalutamide block proliferation of VCaP cells in response to 0.1 nM of the AR agonist R1881, enzalutamide lost antiproliferative potency with increasing R1881 concentrations, whereas ARV-330 maintained antiproliferative effects. In cells containing the ARF876L mutation, enzalutamide was ineffective; however, ARV-330 maintained complete effectiveness. In mice, ARV-330 exhibited good pharmacokinetic properties, with t1/2 values of several hours and bioavailability of >80% after sc injection. Treatment of mice with ARV-330, at doses ranging from 0.3 to 10 mg/kg, resulted in reduction of AR protein levels and prostate involution in normal mice and, in mice implanted with VCaP tumors, reduction in plasma PSA and blockade of tumor growth.

In summary, the AR PROTAC ARV-330 removes AR from prostate cancer cells in a potent manner and produces therapeutic effects as a result. This cellular efficacy has translated into biomarker activity and efficacy in animal models, and ARV-330 is now in preclinical development. Thus, targeted degradation of AR may provide a novel mechanism for providing efficacious therapy for patients with prostate cancer for whom current therapies have failed.

Citation Format: James D. Winkler, Meizhong Jin, Andy P. Crew, AnnMarie K. Rossi, Ryan R. Willard, Hanqing Dong, Kam Siu, Jing Wang, Deborah A. Gordon, Xin Chen, Caterina Ferraro, Craig M. Crews, Kevin Coleman, Taavi K. Neklesa. ARV-330: An androgen receptor PROTAC degrader for prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PR08.
Abstract A14: Loss of LKB1 gene alone causes malignant transformation of astrocytes
Authors: Braun David , Feinstein Douglas , Engelhard Herbert
Published: May 2016

Liver Kinase B1 (LKB1) is a master kinase regulating activation of multiple downstream kinases, and plays roles in cell polarity, metabolism, proliferation, and inflammatory activation. The role LKB1 plays in the malignant transformation of normal astrocytes has not previously been reported. We hypothesized that LKB1 deficiency would influence the growth and differentiation of astrocytes. To test this, we prepared primary cortical astrocytes from homozygous LKB1 f/f mice. When the cells reached confluency, they were infected with AAV-CMV-Cre-GFP virus (knocking out the LKB1 gene), or with AAV-CMV-GFP as control. After 7 additional days, the control cells continued to have the typical appearance of astrocytes, while the LKB1 knockout cells began to form spheroids, with a 5-fold increase in proliferation by MTS assay. The LKB1 knockout astrocytes also showed loss of contact inhibition, increased migrative capability (by scratch assay), and loss of GFAP staining. Our data show that LKB1 deficiency alone induces a transformed phenotype in astrocytes, providing a new model for glioma and indicating its pathogenesis.

Citation Format: David Braun, Douglas Feinstein, Herbert Engelhard. Loss of LKB1 gene alone causes malignant transformation of astrocytes. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A14.
Abstract A24: Brain tumor induction after somatic inactivation of rb1 in zebrafish
Authors: Solin Staci L. , Shive Heather R. , Essner Jeffrey J. , McGrail Maura
Published: May 2016

Purpose: We found that targeted somatic inactivation of rb1 leads to solid tumor formation in the central nervous system of adult zebrafish. The purpose of our study was to characterize the histological and genetic features of the tumors.

Methods: Zebrafish rb1 tumor suppressor was inactivated in somatic tissue by injection of transcription activator-like effector nucleases (TALENs) targeting exon 2 and exon 3 at the one-cell stage. Histopathology and immunohistochemistry with s100 and pH3 was performed on tumor tissue.

To confirm TALEN-mediated editing of the targeted rb1 loci and characterize the frequency and type of indel alleles in tumor and somatic tissues we sequenced cloned amplicons from each locus.

Results: Zebrafish injected with TALENs targeting exon 2 or exon 3 of rb1 presented with cranial tumors beginning at six months of age. By one year the frequency of adults with tumors was 33-50% and 11%, respectively. Histopathological analyses of tumors from eight exon 2-targeted and six exon 3-targeted fish revealed a range of undifferentiated and differentiated tumor phenotypes. Seven tumors exhibited features suggestive of primitive neuroectodermal tumors (PNET). The remainder exhibited more differentiated tumor phenotypes resembling glioma. S100 was detected in a subset of the tumors. pH3 labeled regions of high mitotic activity in each of the tumors. In 4 out of 4 tumors the frequency of rb1 mutant alleles (61%-87% of cloned amplicons) was consistently higher than wild type. However, only one or two specific rb1 alleles were present in 3 out of 4 tumors. In contrast, the mutation frequency in retinal, muscle, and germline tissues was highly variable (3%-100%), and represented by up to seven different mutant rb1 alleles.

Conclusion: Somatic inactivation of rb1 resulted in brain tumors showing varying degrees of differentiation suggesting the possibility that transformed neural progenitors give rise to tumors. rb1 allele frequency is consistent with clonal expansion contributing to tumor growth. Predisposing zebrafish to brain tumors by somatic inactivation of rb1 with site-specific nucleases suggests a simple method for investigating pathways cooperating in tumor progression.

Citation Format: Staci L. Solin, Heather R. Shive, Jeffrey J. Essner, Maura McGrail. Brain tumor induction after somatic inactivation of rb1 in zebrafish. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A24.
Abstract B14: Targeting a network of cancer control nodes through rescue of c-Cbl: A novel therapeutic approach in GBM
Authors: Stripay Jennifer L. , Stevens Brett M. , Bardin Addie L. , Noble Mark D.
Published: May 2016

Glioblastoma multiforme is the most common primary brain tumor in adults, the most malignant of all intracranial tumors, and is associated with inevitable recurrence and 15 month mean survival despite multimodal therapy. The majority of GBM tumors maintain heterogeneous amplifications (many in the absence of genetic mutation) in receptor tyrosine kinases (RTKs), which are mediators of growth factor signaling. Our lab has discovered that control over RTK levels in normal CNS progenitors is maintained by the Redox/Fyn/c-Cbl (RFC) pathway whereby increased oxidative state results in activation of the E3 ubiquitin ligase c-Cbl and the subsequent internalization and degradation of target RTKs. RFC signaling is disrupted in GBM due to a physical sequestration of c-Cbl, preventing pro-oxidative chemotherapeutics from reducing pro-survival signaling through Epidermal Growth Factor Receptor, Platelet-Derived Growth Factor Receptor, etc. Using a triangulating drug screen, we identified candidate FDA-approved small molecules capable of reducing signaling downstream of c-Cbl-targeted RTKs. We found that candidate URMC-06, an antidepressant with a good clinical safety profile, could rescue RFC signaling and significantly enhance the sensitivity of GBM cells to a pro-oxidative agent (Tamoxifen) in a c-Cbl-dependent manner. There is significant evidence to suggest that a tumor initiating cell population mediates the malignancy of GBM tumors. These cells express stem-cell associated markers and exhibit self-renewal capacity. We observed a c-Cbl-dependent inhibition of neurosphere formation (an in vitro measure of self-renewal) and a reduction in the expression of stem cell markers Sox2, beta-catenin, FoxM1 and Nestin in GBM cells treated with URMC-06. Our candidate also facilitated reduction in heat shock signaling and an inhibition of autophagy (both putative survival mechanisms) in GBM cells. URMC-06 was effective, in combination with clinically relevant agents (Temozolomide and Tamoxifen), at reducing tumor progression in an in vivo xenograft model of GBM. Our work supports the hypothesis that c-Cbl is a critical player in GBM that integrates multiple nodes of cancer control and that pharmacological rescue of c-Cbl represents a potentially powerful therapeutic approach.

Citation Format: Jennifer L. Stripay, Brett M. Stevens, Addie L. Bardin, Mark D. Noble. Targeting a network of cancer control nodes through rescue of c-Cbl: A novel therapeutic approach in GBM. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B14.
Abstract B18: Targeting histone deacetylase 6 (HDAC6) depresses proliferation and induces caspase-associated cell death in glioblastoma multiforme (GBM) cells and GBM stem cell-like spheroids
Authors: Saadatzadeh Mohammad Reza , Vishehsaraei Khadijeh Bijangi , Wang Haiyan , Cohen-Gadol Aaron , Pollok Karen E. , Safa Ahmad R.
Published: May 2016

Glioblastoma multiforme (GBM) comprises the most common and very aggressive form of primary brain tumor with a dismal prognosis and very poor response to the current therapies. HDAC inhibitors (HDACi) are capable of inducing growth arrest and apoptosis in various tumor cell types. HDAC6 is a unique enzyme having two deacetylase domains, and a ubiquitin-binding domain. HDAC6 interacts with a number of proteins in the cytoplasm and is involved in tumorigenesis, cell motility, and metastasis. Significantly, it has been shown that HDAC6 knockout mice are viable. Therefore, in this study, we evaluated HDAC6 as a relevant target for GBM treatment by using a potent HDAC6 inhibitor, CAY 10603. Our data showed that CAY 10603 targets the established GBM cell line U86MG, M-HBT161 early primary cultures, and CD133- and SOX2-positive stem cell-like spheroids. CAY 10603 at 1-5 μM triggered significant inhibition of cell survival and induced apoptotic cell death in GBM cells and GBM spheroids. CAY 10603 reduced cell survival in these cells by 50% (IC50) at 1-3 μM treatment for 48 h. Similarly, CAY 10603 induced significant dose-dependent inhibition of spheroid survival in U87MG and M-HBT161 when the spheroids were grown in a defined GBM stem cell (GSC) growth medium on ultra-low attachment plates for four days. Interestingly, when grown in the same medium but on plates which promote attachment, the spheroids were 3-4 fold more sensitive to CAY 10603, indicating that growth conditions affect the sensitivity of GBM stem cell-like spheroids. Moreover, CAY 10603 triggered cell death in U87MG and M-HBT161 cells as well as CD133/SOX2-expressing spheroids was associated with activation of caspases-3, -6 and -9. Overall, our results show that CAY 10603 robustly inhibits the growth of GBM cells, and is effective in eliminating spheroids that contain GBM cancer stem cells which play a major role in drug resistance and disease recurrence. These results suggest that use of CAY 10603 alone or in combination with other agents may potentially improve the survival of brain tumor patients.

Citation Format: Mohammad Reza Saadatzadeh, Khadijeh Bijangi Vishehsaraei, Haiyan Wang, Aaron Cohen-Gadol, Karen E. Pollok, Ahmad R. Safa. Targeting histone deacetylase 6 (HDAC6) depresses proliferation and induces caspase-associated cell death in glioblastoma multiforme (GBM) cells and GBM stem cell-like spheroids. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B18.
Abstract B44: Acquisition of meiotic DNA repair regulators maintain genome stability in glioblastoma
Authors: Rivera Maricruz , Wu Qiulian , Hamerlik Petra , Hjelmeland Anita , Bao Shideng , Rich Jeremy
Published: May 2016

Glioblastoma (GBM), the most prevalent type of primary intrinsic brain cancer in adults, remains universally fatal despite maximal therapy, including radiotherapy and chemotherapy. Cytotoxic therapy generates double stranded DNA breaks (DSBs), most commonly repaired by homologous recombination (HR). We hypothesized that cancer cells coopt meiotic repair machinery as DSBs are generated during meiosis and repaired by molecular complexes distinct from genotoxic responses in somatic tissues. Indeed, we found that gliomas express meiotic repair genes and their expression informed poor prognosis. We interrogated the function of Disrupted Meiotic cDNA1 (DMC1), a homolog of RADiation sensitive51 (RAD51), the primary recombinase used in mitotic cells to search and recombine with the homologous DNA template. DMC1, whose only known function is as an HR recombinase, was expressed by GBM cells and induced by radiation. While targeting DMC1 in non-neoplastic cells minimally altered cell growth, DMC1 depletion in GBM cells decreased proliferation, induced activation of Checkpoint kinase 1 (CHK1) and expression of p21CIP1/WAF1, and increased replication protein A (RPA) foci, suggesting increased replication stress. Combining loss of DMC1 with ionizing radiation (IR) inhibited activation of DNA damage responses and increased radiosensitivity. Furthermore, loss of DMC1 reduced tumor growth and prolonged survival in vivo. Our results suggest that cancers coopt meiotic genes to augment survival under genotoxic stress, offering molecular targets with high therapeutic indices.

Citation Format: Maricruz Rivera, Qiulian Wu, Petra Hamerlik, Anita Hjelmeland, Shideng Bao, Jeremy Rich. Acquisition of meiotic DNA repair regulators maintain genome stability in glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B44.
Abstract IA04: Understanding tumor heterogeneity in glioblastoma
Authors: Tavaré Simon , Sottoriva Andrea , Piccirillo Sara , Spiteri Inma , Touloumis Anestis , Marioni John C. , Curtis Christina N. , Watts Colin
Published: May 2016

Glioblastoma (GB) is advanced at the time of clinical presentation and response to treatment may be confounded by clonal heterogeneity and differential patterns of resistance in spatially distinct parts of the same tumor. However, we do not understand how this complex environment evolves. Accumulating evidence suggests that intra-tumor heterogeneity is likely to be the key to understanding treatment failure, however the dynamics of such heterogeneity are still poorly understood.

Using a Fluorescence-Guided Multiple Sampling technique we obtained samples from the tumor mass, the sub-ependymal zone and the non-fluorescent tumor margin. We performed an integrated genomic analysis to develop a spatial reconstruction of tumor evolution in individual patients. Temporal analysis was performed using patient-derived xenografts obtained by serial transplantation of tumor cells in immuno-suppressed mice.

Phylogenetic reconstruction of the fragments from each GB identifies copy number alterations in EGFR and CDKN2A/B/p14ARF as early evolutionary events while aberrations in PDGFRA and PTEN occur later in the disease progression. Transcriptional profiling reveals that many patients display multiple GB subtypes within their tumor while deconstruction of the clonal organization of each tumor fragment at single-molecule level identified multiple coexisting cell lineages. Ancestral tumor precursors that gave rise to the tumor mass were found in the sub-ependymal zone of a subset of GB patients. When used in in vivo experiments, tumor cells show variable competitive capacity for disease propagation and further genetic diversification, suggesting that GB evolves through complex dynamics of sub-clonal fitness advantage and acquisitions of mutations and copy number alterations.

Our integrated genomic analysis addresses the evolution of GB in individual patients across multiple spatial scales. Our data reveal early clonal diversification generating a genetically complex and highly evolved disease environment at clinical presentation. We propose that these fundamental patient-specific tumor evolutionary dynamics underlie clinical phenotypic heterogeneity and may have implications for the emergence of resistant disease.

Citation Format: Simon Tavaré, Andrea Sottoriva, Sara Piccirillo, Inma Spiteri, Anestis Touloumis, John C. Marioni, Christina N. Curtis, Colin Watts. Understanding tumor heterogeneity in glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr IA04.
Abstract IA17: Genome-epigenome interplay in medulloblastoma
Authors: Northcott Paul A.
Published: May 2016

The recent application of genomics to large series of primary human medulloblastomas has dramatically improved our perspective of the genes, pathways, and molecular processes underpinning this highly malignant pediatric brain tumor. Transcriptionally defined molecular subgroups have emerged, changing the fundamental definition of medulloblastoma from a single histologically recognized entity, to four distinct subtypes: WNT, SHH, Group3, and Group4. These subgroups exhibit highly discriminatory genomic landscapes, patient demographics, and clinical phenotypes. Next-generation sequencing has identified and implicated recurrently altered somatic drivers, many of which are affected in a subgroup-restricted manner. In contrast, somatic variants affecting chromatin-modifying genes, including histone “writers,” “erasers,” and “readers” are prevalent in medulloblastoma and occur across subgroups, suggesting that deregulation of the epigenome is an essential step during medulloblastoma development. Integrative “omics”-based strategies have begun to dissect the fascinating genome-epigenome interplay underlying medulloblastoma, revealing previously undisclosed oncogenic mechanisms and drivers of disease. Furthermore, new insights into the biology and putative origins of the poorly understood Group3 and Group4 medulloblastomas have likewise been revealed. This talk will summarize these recent discoveries afforded through genomics and discuss their potential implications, both in the lab and in the clinic, going forward.

Citation Format: Paul A. Northcott. Genome-epigenome interplay in medulloblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr IA17.
Abstract A03: Krüppel-like Transcription Factor-10 (KLF10) suppresses the TGFβ-induced epithelial-to-mesenchymal transition
Authors: Mishra Vivek Kumar , Kari Vijayalakshmi , Subramaniam Malayannan , Baumgart Simon J. , Nagarajan Sankari , Wegwitz Florian , Spelsberg Thomas C. , Hawse John R. , Johnsen Steven A.
Published: May 2016

The Transforming Growth Factor-β (TGFβ)/SMAD signaling pathway can function as either a tumor suppressor or metastasis promoter during tumor progression. In normal epithelial cells and early stages of epithelial tumorigenesis TGFβ functions as a tumor suppressor to decrease cell proliferation or induce apoptosis. However, during malignant progression tumor cells no longer respond to the anti-proliferative effects of TGFβ, but instead undergo an epithelial-to-mesenchymal transition (EMT) whereby cells acquire a migratory and invasive phenotype which promotes tumor metastasis. Resolution of the dichotomy in TGFβ function and a further understanding of its tumor suppressor and metastasis promoting functions may uncover new strategies for the treatment of epithelial cancers. In previous studies we demonstrated an important role of the TGFβ-Inducible Early Gene-1 (TIEG1)/Krüppel-like Factor-10 (KLF10) as a central regulator of TGFβ/SMAD signaling and the anti-proliferative functions of TGFβ. In this study we examined the role of KLF10 in controlling the TGFβ-induced EMT in human lung adenocarcinoma cells. We show that depletion of KLF10 results in a more pronounced induction of EMT in human A549 lung adenocarcinoma cells as assessed by quantitative real-time PCR, changes in cellular morphology and immunofluorescence microscopy, and transcriptome-wide (RNA-seq) analyses. Moreover, chromatin immunoprecipitation (ChIP) and chromatin immunoprecipitation-sequencing (ChIP-seq) analysis shows that KLF10 directly binds to GC-rich sequences in the promoter region of the EMT-promoting transcription factor SLUG/SNAI2 to repress its transcription. Consistent with these findings, an analysis of KLF10 and SNAI2 expression in lung cancer revealed that while KLF10 levels are decreased in lung cancer vs. normal samples, SNAI2 levels are increased. Additional ChIP studies showed that KLF10 recruits HDAC1 to the SNAI2 promoter and is required for the removal of activating histone acetylation marks. These findings reveal a previously unknown function of KLF10 in suppressing TGFβ-induced EMT and represent a significant advancement in the understanding the molecular dichotomy of TGFβ function during tumor progression.

Citation Format: Vivek Kumar Mishra, Vijayalakshmi Kari, Malayannan Subramaniam, Simon J. Baumgart, Sankari Nagarajan, Florian Wegwitz, Thomas C. Spelsberg, John R. Hawse, Steven A. Johnsen. Krüppel-like Transcription Factor-10 (KLF10) suppresses the TGFβ-induced epithelial-to-mesenchymal transition. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A03.
Abstract A04: Modulation of SAGA mediated nucleosome acetylation by linker DNA and activator
Authors: Mittal Chitvan
Published: May 2016

The SAGA family of co-activators are highly conserved eukaryotic protein complexes that play a major role in regulating gene expression. These complexes facilitate transcription of inducible genes, in part, by acetylating the histone H3 tails of nucleosomes. While the factors that influence nucleosome acetylation are qualitatively understood, quantitative approaches offer the potential to better understand how the interaction between complex nucleosomal substrates, a multi-subunit SAGA complex, and regulatory proteins, combine to remodel the chromatin landscape of gene promoters. Toward this end, we have developed an initial rate, steady-state, acetylation assay using immobilized nucleosomes. For a minimal 147 bp mononucleosome, we find that acetylation activity saturates at a low nucleosome concentration (50 nM KM), and that turnover is quite slow (0.25 min-1 kcat). Addition of linker DNA to the nucleosome enhances acetylation activity up to 8-fold, and does so both by changing the apparent binding constant (2x decrease in KM) and the turnover step (4x increase in kcat). This dual mechanism of stimulation is recapitulated by addition of free DNA in trans, and we are investing the minimum lengths of DNA required for stimulation, as well as the mechanism of stimulation. When the transcriptional activator Gal4-VP16 becomes bound specifically to a recognition site within the linker DNA, additional stimulation of acetylation activity is observed. We are currently working towards gaining more insights into the mechanistic basis of this effect. The results give a more in depth perspective of how different factors work in conjunction to ultimately tune inducible transcription.

Citation Format: Chitvan Mittal. Modulation of SAGA mediated nucleosome acetylation by linker DNA and activator. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A04.