Biology Contribution
Melanoma Cell Intrinsic GABAA Receptor Enhancement Potentiates Radiation and Immune Checkpoint Inhibitor Response by Promoting Direct and T Cell-Mediated Antitumor Activity

https://doi.org/10.1016/j.ijrobp.2020.10.025Get rights and content

Purpose

Most patients with metastatic melanoma show variable responses to radiation therapy and do not benefit from immune checkpoint inhibitors. Improved strategies for combination therapy that leverage potential benefits from radiation therapy and immune checkpoint inhibitors are critical.

Methods and Materials

We analyzed metastatic melanoma tumors in the TCGA cohort for expression of genes coding for subunits of type A γ-aminobutyric acid (GABA) receptor (GABAAR), a chloride ion channel and major inhibitory neurotransmitter receptor. Electrophysiology was used to determine whether melanoma cells possess intrinsic GABAAR activity. Melanoma cell viability studies were conducted to test whether enhancing GABAAR mediated chloride transport using benzodiazepine-impaired viability. A syngeneic melanoma mouse model was used to assay the effect of benzodiazepine on tumor volume and its ability to potentiate radiation therapy or immunotherapy. Treated tumors were analyzed for changes in gene expression by RNA sequencing and presence of tumor-infiltrating lymphocytes by flow cytometry.

Results

Genes coding for subunits of GABAARs express functional GABAARs in melanoma cells. By enhancing GABAAR-mediated anion transport, benzodiazepines depolarize melanoma cells and impair their viability. In vivo, benzodiazepine alone reduces tumor growth and potentiates radiation therapy and α-PD-L1 antitumor activity. The combination of benzodiazepine, radiation therapy, and α-PD-L1 results in near complete regression of treated tumors and a potent abscopal effect, mediated by increased infiltration of polyfunctional CD8+ T cells. Treated tumors show expression of cytokine–cytokine receptor interactions and overrepresentation of p53 signaling.

Conclusions

This study identifies an antitumor strategy combining radiation and/or an immune checkpoint inhibitor with modulation of GABAARs in melanoma using benzodiazepine.

Introduction

The incidence of melanoma continues to rise, and advanced disease confers a poor prognosis.1 Approximately 50% of melanomas harbor somatic B-raf (BRAF) mutations,2,3 which sensitizes them to treatment with BRAF or BRAF/MEK inhibitor combinations. The first generation of a clinically active BRAF inhibitor produced high systemic objective response rates.4 Subsequent development of next-generation combination therapies with BRAF/MEK inhibitors further improved progression-free survival5, 6, 7; however, the majority of patients acquire resistance to these therapies.8, 9, 10, 11 Recently, a combination of immune checkpoint inhibitors targeting programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) produced an overall response rate of approximately 60% and durable responses in a portion of patients, including those with melanoma brain metastases.12, 13, 14, 15, 16 However, the majority of metastatic melanoma patients do not experience durable responses.

Although significant progress has been made, novel therapeutic strategies to treat BRAF/MEK inhibitor– or immune checkpoint inhibitor–resistant disease are desperately needed. Furthermore, there are unique clinical challenges, such as the presence of melanoma brain metastases, which is associated with significant morbidity and mortality. In addition, the brain could represent a therapeutic sanctuary site because of the blood–brain barrier.

Gene expression analysis of melanoma tumors by The Cancer Genome Atlas (TCGA) revealed that GABR genes, which code for subunits of the type A γ-aminobutyric acid (GABA) neurotransmitter receptor (GABAAR), are among those most highly expressed.17 We conducted a more extensive analysis of GABR expression in metastatic melanoma tumors and characterization of melanoma cells for intrinsic GABAAR activity. We find that among melanoma patients, expression of GABR genes varied with the molecular subgroups of melanoma defined by TCGA, including MITF-low and Keratin groups, and that melanoma cells express functional GABAARs. Furthermore, enhanced GABAAR mediated membrane permeability to anions with GABAAR subtype-preferring benzodiazepines, resulting in depolarized mitochondria in melanoma cells and impaired cell viability. In a syngeneic melanoma mouse model, benzodiazepine alone reduces tumor growth; it also potentiates effectiveness when combined with radiation and/or α-PD-L1. Benzodiazepine with radiation promotes both ipsilateral and abscopal antitumor activity associated with increased tumor infiltration with antigen-specific polyfunctional CD8 T cells. Our study identifies a potential novel antitumor strategy combining radiation and/or immune checkpoint inhibitor with modulation of GABAARs in melanoma using a benzodiazepine.

Section snippets

Cell lines

Cell lines were purchased from American Type Culture Collection (ATCC). B16F10 was transduced with lentiviral vector expressing lymphocytic choriomeningitis glycoprotein (GP).

Electrophysiology

Intracellular saline solution contained 145 mM CsCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM EGTA, and 10 mM HEPES. Extracellular saline solution contained 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM D-glucose. Both were adjusted to a pH of 7.4 (320-330 mOsm) and sterilized with a 0.2 μm filter. A375 and

GABAAR subunit expression in metastatic melanoma patient tumors

GABAARs form pentameric chloride channels, composed most commonly of 2 α, 2 β, and γ subunits encoded by GABR genes GABRA (1-6), GABRB (1-3), and GABRG (1-3), respectively (Fig. 1A).29,30 We conducted a differential expression analysis of normal human melanocytes and metastatic melanoma patient-derived lines (Fig. E1). This analysis highlights a difference in expression between melanocytes and melanoma cells. Specifically, there is significantly enhanced expression in the melanoma lines,

Discussion

We show that melanoma lines express GABAAR whose activity is activated by its ligand/agonist GABA and enhanced by specific benzodiazepines. This is consistent with the GABAAR forming a hetero-pentameric structure with a canonical αβαβγ subunit stoichiometry. We show that benzodiazepine QH-II-066 is capable of directly impairing melanoma cell viability and reducing tumor volume in a syngeneic melanoma model, even at a dose comparable to that administered for adult patients for a wide range of

Conclusions

Future experimental effort will need to address whether QH-II-066 acts in synergy with radiation and/or immune checkpoint inhibitor or whether its effect is additive. In addition, how QH-II-066 potentiates radiation, whether its potentiation of radiation is the same as how it might potentiate immune checkpoint inhibitor, and whether the effect of QH-II-066 is related to creating significant oxidative stress in the melanoma cells remain open questions. The results of this study suggest that the

Acknowledgments

The authors thank Drs. Peter Stambrook, Martha Neagu, and Jess Guarnaschelli for helpful comments on the manuscript and Dr. Zalfa Abdel-Malek for providing a primary melanocyte cell line and technical advice for its use in our studies.

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    Daniel A. Pomeranz Krummel and Tahseen H. Nasti are co-first authors.

    This work was supported by NIH-NINDS award number K08-NS083626 (to S.S.); the Thomas E. & Pamela M. Mischell Family Foundation (to S.S.); the Harold C. Schott Foundation funding of the Harold C. Schott Endowed Chair, UC College of Medicine (S.S.); Winship Cancer Institute Melanoma Philanthropic Funds (to S.S. and M.K.K.); NIH-NINDS award number NS089719 (to A.J.); NIH award numbers DA043204 (to Rowlett, James K.) and NS076517 (to J.M.C.); the NSF Division of Chemistry (CHE1625735; to J.M.C.); the Milwaukee Institute for Drug Discovery (to J.M.C.); American Cancer Society Institutional Research Grant (to M.K.K.); the Department of Radiation Oncology, Emory University Research Funds (to M.K.K.); the Merck Sharp & Dohme Corporation (to M.K.K.); NIH-NCI grants K08-CA222663 and NIH-NCI U54-CA225088 (to B.I.); an SITC-BMS Cancer Immunotherapy Translational Fellowship (to B.I.); the Burroughs Wellcome Fund Career Award for Medical Scientists (to B.I.); the Department of Oncology, LIVESTRONG Cancer Institutes, Dell Medical School, University of Texas at Austin, and research funds (to J.K.); CPRIT Scholar Award #RR160093 (to S. Gail Eckhardt); support for the use of analytical instrumentation provided by UW-Milwaukee’s Shimadzu Laboratory for Advanced Applied and Analytical Chemistry (to J.M.C.). Research was also supported in part by the Biostatistics & Bioinformatics and the Integrated Cellular Imaging Shared Resources of the Winship Cancer Institute of Emory University and NIH/NCI under award number P30CA138292. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

    Disclosures: M.K.K. reports a grant from Merck Pharmaceuticals outside the submitted work. S.S. reports an advisory role for NovoCure Ltd. B.I. reports receiving personal fees from Merck and Volastra Therapeutics outside the submitted work. D.A.P.K., J.M.C., and S.S. are cofounders of Amlal Pharmaceuticals Inc. and serve on its board of directors. T.N. reports a patent pending for benzodiazepine-mediated radiosensitization of metastatic melanoma. J.C. reports a patent pending for International PCT Application No. PCT/US2020/034169. S.S., J.C., D.A.P.K. reports a patent pending for International PCT Application No. PCT/US19109. M.K. has applied for provision patent pending.

    The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE159829 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE159829).

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