CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY Venetoclax Cooperates with Ionizing Radiation to Attenuate Diffuse Midline Glioma Tumor Growth Krishna Madhavan 1 , Ilango Balakrishnan 1 , Senthilnath Lakshmanachetty 1 , Angela Pierce 1 , Bridget Sanford 1 , Susan Fosmire 1 , Hanan B. Elajaili 2 , Faye Walker 1 , Dong Wang 1 , Eva S. Nozik 2 , Siddhartha S. Mitra 1,3 , Nathan A. Dahl 1,3 , Rajeev Vibhakar 1,3 , and Sujatha Venkataraman 1,3 ABSTRACT ◥ Purpose: Tumor relapse after radiotherapy is a major hurdle in treating pediatric H3K27M-mutant diffuse midline gliomas (DMG). Radiotherapy-induced stress increases association of BCL2 family of proteins with BH3 pro-apoptotic activators preventing apoptosis. We hypothesized that inhibition of radiotherapy-induced BCL2 with a clinically relevant inhibitor, venetoclax, will block BCL2 activity leading to increased apoptosis. BCL2 has never been implicated in DMG as a radiotherapy-induced resistant mechanism. Experimental Design: We performed an integrated genomic analysis to determine genes responsible for radioresistance and a targeted drug screen to identify drugs that synergize with radiation in DMG. Effect of venetoclax on radiation-na € ve and 6 Gy radiation on cells was evaluated by studying cell death, changes in BCL2 phosphorylation, reactive oxygen species (ROS), and apoptosis, as well as BCL2 association with BH3 apoptosis initiators. The efficacy of combining venetoclax with radiation was evaluated in vivo using orthotopic xenograft models. Results: BCL2 was identified as a key regulator of tumor growth after radiation in DMGs. Radiation sensitizes DMGs to venetoclax treatment independent of p53 status. Venetoclax as a monotherapy was not cytotoxic to DMG cells. Postradiation venetoclax treatment significantly increased cell death, reduced BCL2–BIM association, and augmented mitochondrial ROS leading to increased apoptosis. Combining venetoclax with radiotherapy significantly enhanced the survival of mice with DMG tumors. Conclusions: This study shows that venetoclax impedes the antiapoptotic function of radiation-induced BCL2 in DMG, leading to increased apoptosis. Results from these preclinical studies dem- onstrate the potential use of the BCL2 inhibitor venetoclax com- bined with radiotherapy for pediatric DMG. Introduction Diffuse midline gliomas (DMG) are highly aggressive brain tumors that are uniformly fatal in children (1, 2). The majority of these tumors is present in the brain stem/pons and is classified as diffuse intrinsic pontine glioma (DIPG; refs. 1, 2). As about 5% of this tumor can also be found in other midline structures like thalamus and spinal cord, these tumors are classified together as DMGs (3). Recent studies have shown that a somatic p.Lys27Met substitution in histone-3 (H3.1/H3.3K27M mutation) occurs in more than 85% of patients with DMG and is associated with poor survival (4). The median survival of patients with DMG is about 11 months (5). Given their anatomic location, surgical resection is not possible (6). Fractionated radiotherapy, typically 54–60 Gy over a 6-week period, remains the only standard treatment for DMG (6). Attempts to combine systemic treatments with radiotherapy have been unsuccessful (7). While radiotherapy provides clear clinical benefit, the tumors invariably relapse and can become refractory to other therapies (8). Therefore, identifying mechanisms by which DMG tumor cells become resistant to radiotherapy-induced cytotoxicity is critical in improving the survival of these patients. Ionizing radiation (IR) primarily acts by directly or indirectly enhancing DNA damage and may thereby lead to cell death based on the cell type and the IR dose (9). Oncogenic mutations in many cancers impair apoptosis pathways and cell death mechanisms that promote resistance to radiation (10). Researchers have previously shown that different radiosensitizers increase the effect of radiation on DMG cell survival (11–14). Furthermore, studies have shown that the intrinsic apoptosis pathway in mitochondria, controlled by B-cell lymphoma 2 (BCL2) family, is stimulated in response to radiotherapy in certain tumor types (15). The BCL2 family consists of proapoptotic members (BAX, BAK), antiapoptotic “prosurvival” members (BCL2, MCL1, BCL-xL), and BH3 apoptosis initiators (BIM, BAD; ref. 15). In the typical response to DNA damage, the proapoptotic members BAX and BAK form an oligomer thereby releasing cytochrome c, which is in turn responsible for the activation of caspases, leading to apoptosis and eventually cell death (16). In cancer cells with anomalous expression of the BCL2 family genes, antiapoptotic members bind to proapoptotic members and BH3 apoptosis initiators, blocking the activation of apoptosis (17). The antiapoptotic members are responsible for deac- tivating mitochondrial apoptosis pathways, thereby giving the tumor cell a clear survival advantage following radiation (18). Overexpression of BCL2 family proteins has been well-described in many cancers, but their functional contribution to radioresistance, particularly in DMG is unknown. Pharmacologic inhibition of BCL2 family proteins using various BH3 mimetics has been shown to abrogate radioresistance and induce 1 Morgan Adams Foundation Pediatric Brain Tumor Research Program, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado. 2 Cardiovascular Pulmonary Research Laboratories and Pediatric Critical Care Medicine, Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, Colorado. 3 Department of Pediatrics and Section of Pediatric Hematology/Oncology/BMT, Children’s Hospital Colorado and University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). K. Madhavan, I. Balakrishnan, and S. Lakshmanachetty contributed equally to this article. Corresponding Author: Sujatha Venkataraman, Department of Pediatrics (Hematology-Oncology), University of Colorado Denver, 12800 East 19th Avenue, Aurora, CO 80045. Phone: 303-724-2230; Fax: 303-724-4015; E-mail: Sujatha.Venkataraman@cuanschutz.edu Clin Cancer Res 2022;28:2409–24 doi: 10.1158/1078-0432.CCR-21-4002 Ó2022 American Association for Cancer Research AACRJournals.org | 2409 Downloaded from http://aacrjournals.org/clincancerres/article-pdf/28/11/2409/3149801/2409.pdf by guest on 14 June 2023