cancers Article Targeted Mass Spectrometry Enables Quantification of Novel Pharmacodynamic Biomarkers of ATM Kinase Inhibition Jeffrey R. Whiteaker 1 , Tao Wang 1, Lei Zhao 1, Regine M. Schoenherr 1, Jacob J. Kennedy 1, Ulianna Voytovich 1, Richard G. Ivey 1, Dongqing Huang 1, Chenwei Lin 1, Simona Colantonio 2, Tessa W. Caceres 2, Rhonda R. Roberts 2, Joseph G. Knotts 2, Jan A. Kaczmarczyk 2, Josip Blonder 2, Joshua J. Reading 2, Christopher W. Richardson 2, Stephen M. Hewitt 3, Sandra S. Garcia-Buntley 2, William Bocik 2, Tara Hiltke 4, Henry Rodriguez 4, Elizabeth A. Harrington 5, J. Carl Barrett 5, Benedetta Lombardi 5, Paola Marco-Casanova 5, Andrew J. Pierce 5 and Amanda G. Paulovich 1,* 1 Fred Hutchinson Cancer Research Center, Clinical Research Division, Seattle, WA 98109, USA; [email protected] (J.R.W.); [email protected] (T.W.); [email protected] (L.Z.); [email protected] (R.M.S.); [email protected] (J.J.K.); [email protected] (U.V.); [email protected] (R.G.I.); [email protected] (D.H.); [email protected] (C.L.) 2 Cancer Research Technology Program, Antibody Characterization Lab, Frederick National Laboratory for Cancer Research, Frederick, MD 21701, USA; [email protected] (S.C.); [email protected] (T.W.C.); [email protected] (R.R.R.); [email protected] (J.G.K.); [email protected] (J.A.K.); [email protected] (J.B.); [email protected] (J.J.R.); [email protected] (C.W.R.); [email protected] (S.S.G.-B.); [email protected] (W.B.) 3 Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Citation: Whiteaker, J.R.; Wang, T.; Institute, National Institute of Health, Bethesda, MD 20892, USA; [email protected] Zhao, L.; Schoenherr, R.M.; Kennedy, 4 Office of Cancer Clinical Proteomics Research, National Cancer Institute, Bethesda, MD 20892, USA; J.J.; Voytovich, U.; Ivey, R.G.; Huang, [email protected] (T.H.); [email protected] (H.R.) D.; Lin, C.; Colantonio, S.; et al. 5 Translational Sciences, Oncology, AstraZeneca, Cambridge CB4 0WG, UK; Targeted Mass Spectrometry Enables [email protected] (E.A.H.); [email protected] (J.C.B.); Quantification of Novel [email protected] (B.L.); [email protected] (P.M.-C.); Pharmacodynamic Biomarkers of [email protected] (A.J.P.) * Correspondence: [email protected]; Tel.: +1-206-667-1912 ATM Kinase Inhibition. Cancers 2021, 13, 3843. https://doi.org/10.3390/ cancers13153843 Simple Summary: Functionality of the cellular DNA damage response (DDR) network affects risk for developing cancer, and the DDR is also a target of cancer therapies. Thus, it is important that we have Academic Editors: Udayan Guha and reliable laboratory methods for determining the activity of this network. Wedescribe the development and Xu Zhang analytical validation of a targeted mass spectrometry-based 51-plex assay (DDR-2) for measuring proteins and post-translational modifications related to the DDR. The findings demonstrate identification of Received: 18 June 2021 potential novel pharmacodynamic biomarkers. Accepted: 23 July 2021 Published: 30 July 2021 Abstract: The ATM serine/threonine kinase (HGNC: ATM) is involved in initiation of repair of DNA double-stranded breaks, and ATM inhibitors are currently being tested as anti-cancer agents in Publisher’s Note: MDPI stays neutral clinical trials, where pharmacodynamic (PD) assays are crucial to help guide dose and scheduling with regard to jurisdictional claims in and support mechanism of action studies. To identify and quantify PD biomarkers of ATM inhibition, published maps and institutional affil- we developed and analytically validated a 51-plex assay (DDR-2) quantifying protein expression and iations. DNA damage-responsive phosphorylation. The median lower limit of quantification was 1.28 fmol, the linear range was over 3 orders of magnitude, the median inter-assay variability was 11% CV, and 86% of peptides were stable for storage prior to analysis. Use of the assay was demonstrated to quantify signaling following ionizing radiation-induced DNA damage in both immortalized Copyright: © 2021 by the authors. lymphoblast cell lines and primary human peripheral blood mononuclear cells, identifying PD Licensee MDPI, Basel, Switzerland. This article is an open access article biomarkers for ATM inhibition to support preclinical and clinical studies. distributed under the terms and conditions of the Creative Commons Keywords: multiple reaction monitoring; DNA damage response; immuno-MRM; targeted proteomics Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cancers 2021, 13, 3843. https://doi.org/10.3390/cancers13153843 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 3843 2 of 25 1. Introduction The DNA damage response (DDR) is a highly regulated signal transduction network that orchestrates the temporal and spatial organization of protein complexes required to detect and repair (or tolerate) DNA damage (e.g., nucleotide excision repair, base excision repair, homologous recombination, non-homologous end joining, post-replication repair) [1]. The DDR is of critical significance in oncology, as genetic instability caused by dysregulation of the DDR is a key feature of cancer cells [2], and chemotherapy and radiation therapy induce DNA damage to promote cell death. Thus, components of the DDR network are potential therapeutic targets, and being able to quantify and understand activity in the DDR network has clinical implications for cancer patients and those at risk [3]. The Ataxia-telangiectasia-mutated (ATM) gene encodes a serine/threonine protein kinase that initiates the DDR to DNA double-stranded breaks (DSB). ATM is activated through dissociation of the ATM homodimer to active monomers via autophosphorylation at several sites [4], including Ser1981 [5], Ser367 [6], and Ser2996 [7]. Activated ATM phosphorylates downstream components to mediate DNA repair and cell cycle regulation. Cells lacking ATM, as seen in patients with ataxia telangiectasia, are sensitive to DNA damage [8]. Three ATM inhibitors developed by KuDOS (Cambridge, UK) and AstraZeneca (Cambridge, UK), KU-55933, AZD0156, AD1390, display potent and exquisite inhibition of ATM with no meaningful capacity to inhibit other phosphatidylinositol 3-kinase-related kinases (PIKKs), such as ATR and DNA-PK [9–11]. AZD0156 is a particularly potent inhibitor of ATM and is in clinical trials (NCT02588105) in combination with DNA damage- inducing agents, where it is expected to enhance cell death in tumor cells by reducing their capacity to resolve DNA damage. Quantifying proteins and protein networks for pharmacokinetic (PK), pharmaco- dynamic (PD), and proof of mechanism (POM) studies is critical for translating novel therapies, such as ATM inhibitors. Indeed, AstraZeneca recently revised its drug devel- opment framework [12,13] with a view to increasing success rates of drugs in pivotal Phase 3 studies. Key components are POM studies and determining PK/PD relation- ships in early phase clinical trials. Conventional immunoassay platforms (e.g., ELISA, immunohistochemistry) are generally used for these studies; however, these platforms are critically dependent on the absolute specificity of individual antibodies, and establishing this specificity is costly in terms of time and resource. As a result, only a handful of fully validated assays can be developed for each drug project, where the choice of which assays to develop is largely done on the basis of “best educated guess” arising from orthogonal preclinical methods such as Western blotting. Additionally, in many cases an assay for a given target cannot be quickly developed because there are no fit-for-purpose antibodies available. In such cases a bespoke reagent antibody generation campaign may be initiated, adding to the time and cost of drug development. Adding to the issue, proteins act as interconnected “networks”, and the effects of cancer driver mutations spread throughout the network; thus, ideally we would have assays to quantify panels of multiple proteins in early phase clinical trials to assess the activity of pathways/networks that determine treatment responses [14], and this developmental effort is not practical using conventional platforms. Thus, new tools are needed for quantifying proteins and post-translational modifications to aid discovery of new pharmacodynamic markers, enhance mechanistic studies, and translate research findings to clinical implementation. To address this need, we applied targeted multiple reaction monitoring (MRM) mass spectrometry (MS) to develop quantitative assays to proteins and post-translational modifi- cations [15,16]. MRM is a targeted mode of MS in which the full analytical capacity of the instrument is focused on a set of specific analytes, maintaining a high degree of specificity and enhancing the sensitivity of detection [17,18]. By using spiked-in isotopically labeled internal standards, precise relative quantification is achieved, and results can be harmo- nized across laboratories [19,20]. To analyze low abundance proteins and post-translation modifications, including phosphorylation, we coupled an antibody enrichment step to MRM [21]. These “immuno-MRM” assays enable precise quantification of low abundance Cancers 2021, 13, 3843 3 of 25 proteins [22], high multiplexing capability while maintaining specificity for individual analytes [23,24], and