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Inducible Transgene Expression in PDX Models Liu et al. Biomarker Research (2020) 8:46 https://doi.org/10.1186/s40364-020-00226-z RESEARCH Open Access Inducible transgene expression in PDX models in vivo identifies KLF4 as a therapeutic target for B-ALL Wen-Hsin Liu1, Paulina Mrozek-Gorska2, Anna-Katharina Wirth1, Tobias Herold1,3, Larissa Schwarzkopf1, Dagmar Pich2, Kerstin Völse1, M. Camila Melo-Narváez2, Michela Carlet1, Wolfgang Hammerschmidt2,4 and Irmela Jeremias1,5,6* Abstract Background: Clinically relevant methods are not available that prioritize and validate potential therapeutic targets for individual tumors, from the vast amount of tumor descriptive expression data. Methods: We established inducible transgene expression in clinically relevant patient-derived xenograft (PDX) models in vivo to fill this gap. Results: With this technique at hand, we analyzed the role of the transcription factor Krüppel-like factor 4 (KLF4) in B-cell acute lymphoblastic leukemia (B-ALL) PDX models at different disease stages. In competitive preclinical in vivo trials, we found that re-expression of wild type KLF4 reduced the leukemia load in PDX models of B-ALL, with the strongest effects being observed after conventional chemotherapy in minimal residual disease (MRD). A nonfunctional KLF4 mutant had no effect on this model. The re-expression of KLF4 sensitized tumor cells in the PDX model towards systemic chemotherapy in vivo. It is of major translational relevance that azacitidine upregulated KLF4 levels in the PDX model and a KLF4 knockout reduced azacitidine-induced cell death, suggesting that azacitidine can regulate KLF4 re-expression. These results support the application of azacitidine in patients with B-ALL as a therapeutic option to regulate KLF4. Conclusion: Genetic engineering of PDX models allows the examination of the function of dysregulated genes like KLF4 in a highly clinically relevant translational context, and it also enables the selection of therapeutic targets in individual tumors and links their functions to clinically available drugs, which will facilitate personalized treatment in the future. Keywords: PDX models of acute leukemia, Inducible transgene expression, KLF4, Azacitidine * Correspondence: [email protected] 1Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU), Marchioninistraße 25, 81377 Munich, Germany 5German Cancer Consortium (DKTK), Partner Site Munich, Munich, Germany Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Liu et al. Biomarker Research (2020) 8:46 Page 2 of 15 Introduction We developed a tetracycline-inducible expression sys- Tumor cells are characterized by multiple alterations at tem to re-express KLF4 in PDX B-ALL cells in vivo. We mRNA and protein levels. While nongenomic alterations demonstrate here that KLF4 expression reduces tumor are easily identified by techniques such as RNA sequen- growth and enhances the chemotherapeutic response in cing or proteomics [1–3], the functional consequences this tumor model. With the aid of a CRISPR/Cas9-medi- of these alterations are generally uncertain, and only a ated KLF4 knockout in PDX cells, we further demon- small minority of such alterations might play an essential strated that azacitidine exerts its antitumor effect by role in the growth and maintenance of the tumor. Iden- upregulating KLF4, supporting our interpretation. Our tifying essential alterations is of major clinical import- data demonstrate that inducible gene expression in PDX ance because they represent putative targets for models is feasible and can be used to characterize the treatment [1–4]. Clearly, techniques to determine the contribution of selected genes to tumor maintenance functional consequences of alterations are lacking, but and to obtain valuable information regarding therapy re- we established a clinically relevant method to identify al- sponses. Our results reveal that KLF4 is a therapeutic terations with life-sustaining function in individual tu- target of interest in B-ALL, supporting the use of KLF4- mors in vivo. regulating drugs in clinical trials of B-ALL. As a model system, we chose patient-derived xenografts (PDX), which avoids many of the limitations associated Materials and methods with the use of conventional cell line models or primary Ethical statements patient tumor cells [5–7]. PDX faithfully recapitulate the Prior to obtaining the two primary B-ALL patient sam- genetic and phenotypic characteristics of their parental tu- ples (Table S1), written informed consent was obtained mors and their preclinical value for drug testing and bio- from all patients or from parents/caregivers in cases in marker identification is well established [8, 9]. Thus, the which patients were minors. The study was performed use of orthotopic PDX models represent the most clinic- in accordance with the ethical standards of the respon- ally relevant approach to date for studying individual tu- sible committee for human experimentation (written ap- mors [6, 10]. We studied B-cell precursor ALL (B-ALL), proval by the Ethikkommission des Klinikums der the most common malignancy in children which displays Ludwig-Maximilians-Universität Munich, number 068– a favorable prognosis at first diagnosis due to conventional 08 and 222–10) and with the Helsinki Declaration of chemotherapy and, e.g., CAR-T-cell therapy, but harbors 1975, as revised in 2000. an urgent need for better treatment options upon prog- Animal trials were performed in accordance with the nostically challenging disease relapse [11]. current ethical standards of the official committee on To mimic the situation faced by patients, established animal experimentation (written approval by the Regier- tumors should already exist in the preclinical surrogate ung von Oberbayern, [email protected]; animals at the moment of molecular manipulation, July 2010, number 55.2–1-54-2531-95-10; July 2010, which requires the use of inducible in vivo expression 55.2–1-54-2531.6-10-10; January 2016, ROB-55.2 Vet- systems. While these techniques are well established in 2532. Vet_02–15-193; May 2016, ROB-55.2 Vet-2532. most other tumor models and normal human lympho- Vet_02–16-7 and August 2016, ROB-55.2 Vet-2532.Vet_ cytes, PDX models are often excluded from functional 03–16-56). genomic studies [12], and inducible orthotopic PDX models have not been reported. Genetic engineering of EBV We generated an inducible expression system in PDX In the maxi-Epstein Barr virus (EBV) plasmid, wtKLF4 models in mice in vivo. We applied and tested the method and mutKLF4 cDNAs were fused to the 3′ open reading using the signaling molecule and transcription factor KLF4, frame of the viral EBNA2 gene via a T2A element, which which is implicated in the stress-responsive regulation of mediated the coexpression of both genes from the same cell cycle progression, apoptosis and differentiation as well transcript. While the wtKLF4 construct contained the as stemness and pluripotency [13–19]. KLF4 is downregu- entire open reading frame, the mutKLF4 construct lated in numerous cancers but upregulated in others, and lacked the two N-terminal zinc finger domains [40]. De- abnormal KLF4 expression might reflect oncogenic or tails on the generation of both mutant EBV constructs tumorsuppressorfunctionsdepending on the cellular con- are available in the supplement. text, tumor type, subtype and stage [14, 17, 20–27]. KLF4 is frequently deregulated in T-ALL and B-cell tumors [28– Genetic engineering of PDX B-ALL cells for inducible 34], and is reportedly downregulated in pediatric B-ALL transgene expression [35–38]. While KLF4 downregulation has been implicated Primary patient B-ALL cells were transplanted into im- in B-ALL leukemogenesis [39], its functional role in estab- munocompromised mice to generate the PDX models. lished B-ALL cells from patients in vivo is uncertain. PDX B-ALL cells were lentivirally transduced and Liu et al. Biomarker Research (2020) 8:46 Page 3 of 15 transgenic cells were enriched using flow cytometry by to achieve, but also not required, as protein amounts are gating on the recombinantly expressed fluorochromes as calculated with high sensitivity and reliability. described previously [41]. For inducible transgene ex- Primary antibody against KLF4 was purchased from pression, PDX B-ALL cells were consecutively lentivi- R&D systems (AF3640, Minneapolis, MN, USA), against rally transduced with three constructs containing the tet PARP from Cell Signaling Technology (9542 T, USA), activator, the tet repressor and KLF4 expression cas- against GAPDH from R&D systems (MAB5718-SP, settes under the control of the TRE promoter [42]. USA), against Caspase-3 from Millipore (05–654, Biller- ica, USA) and against β-Actin from Novus biologicals In vivo experiments (NB600-501SS, Littleton, USA). A comparison of the re- Leukemia growth and treatment effects were monitored sults obtained using conventional Western Blot versus using bioluminescence in vivo imaging as described pre- the capillary immunoassay is provided in Supplementary viously [41].
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