Mitchell S. Anscher · Kristoffer Valerie Editors Strategies to Enhance the Therapeutic Ratio of Radiation as a Cancer Treatment Strategies to Enhance the Therapeutic Ratio of Radiation as a Cancer Treatment Mitchell S Anscher • Kristoffer Valerie Editors Strategies to Enhance the Therapeutic Ratio of Radiation as a Cancer Treatment Editors Mitchell S Anscher Department of Radiation Oncology Massey Cancer Center Virginia Commonwealth University Richmond, VA, USA Kristoffer Valerie Department of Radiation Oncology Massey Cancer Center Virginia Commonwealth University Richmond, VA, USA ISBN 978-3-319-45592-1 ISBN 978-3-319-45594-5 (eBook) DOI 10.1007/978-3-319-45594-5 Library of Congress Control Number: 2016954313 © Springer International Publishing Switzerland 2016 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made Printed on acid-free paper This Springer imprint is published by Springer Nature The registered company is Springer International Publishing AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Contents Mechanisms of Normal Tissue Response Jolinta Y Lin, Isabel L Jackson, and Zeljko Vujaskovic The Role of Hypoxia in Radiation Response Monica M Olcina, Ryan Kim, and Amato J Giaccia 29 The Role of Cancer Stem Cells in Tumour Radioresponse Annett Linge, Anna Dubrovska, Michael Baumann, and Mechthild Krause 43 Novel Strategies to Prevent, Mitigate or Reverse Radiation Injury and Fibrosis Pierre Montay-Gruel, Gael Boivin, and Marie-Catherine Vozenin 75 Technology Based Strategies to Enhance the Therapeutic Ratio 109 David V Fried and Shiva K Das Nitric Oxide Synthase Uncoupling in Tumor Progression and Cancer Therapy 139 Ross B Mikkelsen, Vasily A Yakovlev, Christopher S Rabender, and Asim Alam Aiming the Immune System to Improve the Antitumor Efficacy of Radiation Therapy 159 Chunqing Guo, Timothy Harris, and Xiang-Yang Wang The Role of MicroRNAs in Modulating Tissue Response to Radiation 183 Rebecca J Boohaker and Bo Xu v Chapter 12 Radiosensitizing Glioma by Targeting ATM with Small Molecule Inhibitors Amrita Sule and Kristoffer Valerie Abstract Malignant glioma is a devastating and incurable brain cancer Current standard treatment of malignant glioma is surgery followed by chemotherapy and radiation Progress during the past few decades in improving long-term survival has been painfully slow with a median overall survival currently at a little more than year New strategies targeting the DNA damage response, including the ATM (ataxia telangiectasia mutated) kinase, are currently being pursued ATM is a master regulator of cell cycle checkpoints, DNA repair, and cell death in response to radiation Pre-clinical studies using novel small molecule inhibitors of the ATM kinase are in progress and results from these look promising for future testing in humans In fact, one ATM kinase inhibitor is currently in a Phase I trial in combination with chemotherapy of advanced solid cancers This chapter focuses on discussing recent advances in developing and testing highly specific inhibitors targeting the ATM kinase for cancer therapy with focus on malignant glioma Keywords Ataxia telangiectasia mutated (ATM) • Convection-enhanced delivery (CED) • DNA damage response (DDR) • Glioblastoma multiforme (GBM) • Ionizing radiation (IR) • Malignant glioma • Mitotic catastrophe • Phosphatidylinositol 3-kinase-related kinase (PIKK) • p53 • Radiosensitizer • Radiotherapy • Temozolomide (TMZ) 12.1 Introduction Nearly 80,000 new cases of malignant glioma (classified by the World Health Organization (WHO) as Grade III and IV glioma) are diagnosed each year in the United States with 17,000 people dying from the disease Grade IV is also referred A Sule • K Valerie (*) Department of Radiation Oncology, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA e-mail: kristoffer.valerie@vcuhealth.org © Springer International Publishing Switzerland 2016 M.S Anscher, K Valerie (eds.), Strategies to Enhance the Therapeutic Ratio of Radiation as a Cancer Treatment, DOI 10.1007/978-3-319-45594-5_12 289 290 A Sule and K Valerie to as glioblastoma multiforme (GBM) GBM is a highly lethal brain tumor presented as one of two subtypes with distinct clinical histories and molecular profiles Hallmark characteristics of GBM include uncontrolled cell proliferation, diffuse infiltration, and resistance to apoptosis These features account for GBM’s poor prognosis and resistance toward radio- and chemotherapy, and a median patient survival of only 12–15 months [1] In older individuals, the most common form is of the primary subtype which arises de novo with no prior symptoms or evidence of progression from low-grade tumors The secondary subtype of GBM occurs in younger patients from lower grade glioma GBMs are classified into four different subgroups based on gene expression profiling; (1) classical, (2) mesenchymal, (3) neural, and (4) pro-neural [2, 3] Primary GBM is mostly found in the classical subgroup with EGFR mutation/amplifications and mutations in CDKN2A and PTEN On the other hand, secondary GBMs are usually found in the pro-neural subgroup with frequent mutations in PDGFR, IDH1/2, and p53 [2] The frequency of p53 mutation in this sub-group is 65 % or greater whereas classical GBM harbors p53 mutations 30 % of the time [4, 5] Recently, a new more reliable molecular classification based on IDH status and specific TERT promoter mutations was proposed [6, 7] Standard treatment of GBM is surgery followed by temozolomide (TMZ), an alkylating drug, and radiation [8, 9] However, little improvement has been seen in the long-term survival of patients with GBM during the last several decades Thus, new treatments and approaches are urgently needed As the understanding of the molecular mechanisms associated with GBM continues to expand, and more specific and potent drugs are developed, efficient delivery of therapeutic agents to the brain becomes very important and remains a challenging clinical problem In particular, both the blood–brain barrier (BBB) and blood–tumor barrier hamper the successful treatment of brain tumors by severely limiting access of therapeutic agents to the brain and tumor [10, 11] These obstacles have made the efficient delivery of anticancer drugs to the brain a major technical hurdle, and therefore this area of research is lagging behind the development of the drugs themselves Because surgery is standard treatment for GBM, the delivery of therapeutic agents directly to the brain during surgery, e.g GLIADEL® wafers, or post-surgery by convection-enhanced delivery (CED) via a cannula and positive pressure does not deviate significantly from current treatment practice For the obvious reasons of being easier to administer and lower cost, an orally bioavailable and BBB-penetrable ATM inhibitor would be preferable over CEDbased delivery However, specific circumstances might favor the latter route of drug administration, e.g if radiomimetic drugs, such as etoposide, and camptothecin, etc., that either are too toxic when administered systemically or are BBBimpermeable, CED could be the most efficacious and appropriate mode of delivery [11] There have been significant advances in the development and pre-clinical testing of radiosensitizers for high grade gliomas during the past few years with focus on targeting the DNA damage response (DDR) (see [12–14] for recent reviews) Despite the identification of exciting new targets and the development of drugs 12 Radiosensitizing Glioma by Targeting ATM with Small Molecule Inhibitors 291 against these targets, their clinical use is still under evaluation One of the earliest targets identified and pursued is the protein mutated in ataxia telangiectasia (ATM) and its intrinsic protein kinase [15] ATM is mutated in the human autosomal recessive disorder, ataxia-telangiectasia (A-T) [16] The extreme radiosensitivity of cells from A-T patients has been known since the 1970s [17] ATM, a serine-threonine kinase and member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, is a major regulator of the DDR ATM is activated in response to DNA double strand breaks (DSBs) induced by DNA damage such as ionizing radiation (IR) or spontaneously during replication and cell growth Once activated, ATM phosphorylates numerous proteins involved in cell cycle regulation, DNA repair, apoptosis, etc [18, 19] ATM-mediated phosphorylation and other subsequent posttranslational modifications affect the stability, sub-cellular localization, and the interaction of proteins involved in these processes, thereby masterminding the DDR [20] ATM is also known to regulate insulin and other growth factor signaling responses resulting from the stimulation with non-classical DDR agents suggesting a much broader role for ATM in regulating cell growth and homeostasis in addition to the DDR [16] During the past 10 years the ATM inhibitor KU-55933 has extensively been used in tissue culture experiments by numerous laboratories to demonstrate the involvement of the ATM kinase in various capacities KU-55933 was developed by KuDOS Pharmaceuticals, Ltd, in the United Kingdom, and shown to be a highly specific ATM kinase inhibitor competitively binding to the ATP-pocket [21] The KU-55933 IC50 for ATM (13 nM) is at least 200-fold lower than for the other PIKKs, including DNA-PKcs and ATR Around 2007, at the time KuDOS was acquired by AstraZeneca, we were offered an improved analog, KU-60019, to test as a radiosensitizer in our mouse glioma models We extensively characterized KU-60019 in vitro with glioma cells to assess its impact on the DDR [22] Briefly, in addition to the improved radiosensitization seen with KU-60019, we documented high specificity toward the ATM kinase with no effect on 229 other kinases in vitro Radiosensitization was observed with all cell lines tested, whether tumor or normal, except for A-T cells, strongly suggesting that the ATM kinase was the target for KU-60019 Furthermore, KU-60019 has high stability and is quickly reversible in vitro in wash out experiments Additionally, we carried out limited in vitro combination testing of KU-60019, temozolomide (TMZ), and radiation [23] When U87 glioma cells were co-treated with KU-60019 and TMZ a slight increase in radiation-induced cell killing was noted although TMZ alone was unable to radiosensitize the cells In addition, without radiation, KU-60019 with or without TMZ reduced glioma cell growth but had no significant effect on the survival of human astrocytes [23] Another study showed a beneficial interaction of KU-55933 and TMZ in vitro but only with inherently TMZ-sensitive glioma cell lines [24] Thus, there is no reason to believe that an ATM kinase inhibitor would be counter-effective with current standard care of glioma Other ATM inhibitors, such as CP466722 [25] and KU-59403 [26], have been developed with only the latter evaluated in a pre-clinical setting and neither one tested against glioma 292 A Sule and K Valerie Fig 12.1 Potential impact of an ATM inhibitor in combination with a DNA damaging agent on cell cycle checkpoints, DNA repair, and cell death The ATM kinase phosphorylates >700 proteins, some at multiple sites, that is necessary for fully triggering the DDR [27] Blocking the DDR including G1 and G2 arrest, DNA repair, and apoptosis/cell death with an ATM inhibitor is expected to affect many cellular responses to radiation and chemotherapy and kill tumor cells Descriptors; →, activation/phosphorylation (Ⓟ); inhibition, ⊥ 12.2 12.2.1 Rationale for Targeting the ATM Kinase Advantages of ATM Kinase-Directed Therapy It was realized early on that an ATM inhibitor would likely serve as an excellent radiosensitizer based on the radiosensitivity of A-T patients [17] The basic idea behind this notion is that an ATM kinase inhibitor, such as KU-55933 or KU-60019, would be expected to block cell cycle checkpoints and DNA repair so that tumor cells would die from apoptosis or other cell death (Fig 12.1) Many proteins regulating cell cycle checkpoints (e.g., p53, MDM2, and CHK2), DNA repair (BRCA1, NBS1), cell death/apoptosis (cABL) are directly phosphorylated by ATM [16, 27, 28], so an ATM inhibitor would effectively block signaling and prevent all downstream DDRassociated processes from taking place with fatal consequences to the tumor cell Cancer-specific targeting is a long-sought-after goal in cancer therapy We demonstrated for the first time that a small molecule ATM kinase inhibitor, KU-60019, efficiently radiosensitized orthotopic gliomas with a much greater response seen with mutant p53 relative to matched glioma with normal p53 [29] Briefly, human glioma U87 cells (p53 wild type) transduced with a retrovirus expressing a p53-281G mutant were grown intra-cranially in nude mice in parallel with mice .. .Strategies to Enhance the Therapeutic Ratio of Radiation as a Cancer Treatment Mitchell S Anscher • Kristoffer Valerie Editors Strategies to Enhance the Therapeutic Ratio of Radiation as a Cancer. .. KINASE KINASE KINASE KINASE KINASE KINASE ATM RAS ? RAF AKT Cytoplasm EGF EGF KINASE KINASE KINASE KINASE KINASE KINASE Plasma membrane INS IGF-1R MEK PP 2A ERK ATM DSB repair/ radioresistance... including the plasma membrane, cytoplasm, and nucleus The observation that both RASRAF-MEK-ERK as well as AKT signaling are affected by ATM manipulation has been reported by a number of laboratories including