Combination Therapy with Radiation and PARP Inhibition Enhances Responsiveness to Anti-PD-1 Therapy in Colorectal Tumor Models

Abstract

Purpose: The majority of colorectal cancers are resistant to cancer immune checkpoint inhibitors. Ionizing radiation (IR) and several radiosensitizers, including PARP inhibitors, can enhance responsiveness to immune checkpoint inhibitors by potentially complementary mechanisms of action. We assessed the ability of radiation and PARP inhibition to induce proimmunogenic changes in tumor cells and enhance their in vivo responsiveness to anti-PD-1 antibodies.

Methods and materials: We performed a candidate drug screen and used flow cytometry to assess effects of the PARP inhibitor veliparib on IR-mediated changes in MHC-1 antigen presentation and surface localization of immune-modulating proteins including PD-L1 and calreticulin in colorectal cancer tumor models. Reverse transcription polymerase chain reaction was used to assess the effects of veliparib and radiation on the expression of proinflammatory and immunosuppressive cytokines. The ability of concurrent PARP inhibition and subablative doses of radiation therapy to enhance in vivo responsiveness to anti-PD-1 antibodies was assessed using unilateral flank-tumor models with or without T-cell depletion.

Results: Veliparib was a potent radiosensitizer in both cell lines. Radiation increased surface localization of MHC-1 and PD-L1 in a dose-dependent manner, and veliparib pretreatment significantly enhanced these effects with high (8 Gy) but not with lower radiation doses. Enhancement of MHC-1 and PD-L1 surface localization by IR and IR+ veliparib remained significant 1, 3, and 7 days after treatment. IR significantly increased delayed tumoral expression of proinflammatory cytokines interferon-Ƴ and CXCL10 but had no significant effect on the expression of IL-6 or TGF-β. Concurrent administration of veliparib and subablative radiation therapy (8 Gy × 2) significantly prolonged anti-PD-1-mediated in vivo tumor growth delay and survival in both tumor models. Moreover, these effects were more pronounced in the microsatellite instability-mutated MC38 tumor model. Enhancement of anti-PD-1 mediated tumor growth delay with veliparib and IR was attenuated by CD8+ T-cell depletion.

Conclusions: We provide preclinical evidence for a novel therapeutic strategy to enhance responsiveness of colorectal tumors to immune checkpoint inhibitors.

Conflict of interest statement

Conflicts of Interest Statement: None of the authors have any conflicts to disclose

Copyright © 2020 Elsevier Inc. All rights reserved.

Figures

Figure 1:. Candidate drug screen.

CT26 (A,C) and MC38 (B,D) cells were treated with varying concentrations of entinostat, IPI-549, VE-821, and veliparib and survival was assessed using clonogenic survival assays to determine IC10 and IC50 values (A-B). Radiosensitization was assessed in both cell lines pretreated with indicated concentrations of each drug and 0–8 Gy of radiation (C-D). Survival was assessed using clonogenic survival assays to determine SER for each drug. N=3–5 for each treatment condition. * indicates p

Figure 2:. Veliparib pretreatment enhances IR-mediated MHC-1 surface localization.

MHC-1 surface localization was assessed using FLOW cytometry in unpermeabilized cells 24 hours after treatment with a single dose of radiation (0–8 Gy) +/− pretreatment with 5 μM veliparib. MFIs for MHC-1 H2-D subunit for each condition in CT26 (A) and MC38 (B) cells. Representative histograms are shown for each cell line (C-D). Expression of antigen processing proteins MHC-1 (H2-D subunit), TAP-1, and β2-macroglobulin was assessed using IR-PCR 24 hours after treatment with 5 μM veliparib, IR (8 Gy), or both (E-F)). Prolonged surface localization of MHC-1 was assessed by FLOW cytometry in unpermeabilized cells 1, 3, and 7 days after treatment (G-H). N=3–5 for each treatment condition. * indicates p

Figure 3:. Veliparib enhances delayed expression of inflammatory cytokines and chemokines.

Expression of INF-β, CXCL10, IL-6, and TGF-β was assessed 1, 3, and 7 days after treatment with veliparib (5 μM), IR (8 Gy) or both and normalized to untreated controls in CT26 (A) and MC38 (B) cells. N=3 for each treatment condition. * indicates P

Figure 4:. Veliparib enhances IR-mediated surface localization of PD-L1.

PD-L1 surface localization was assessed 24 hours after treatment of radiation (0–8 Gy) +/− veliparib (5 μM) pretreatment using FLOW cytometry in unpermeabilized cells (A-B). Prolonged surface localization of PD-L11 was assessed by FLOW cytometry in unpermeabilized cells 72 hours and 1 week after treatment (C-D). Calreticulin surface localization was assessed 24 hours after treatment with drug-free media, veliparib (5 μM), IR (8 Gy), or both (E-F). N=3–5 for each condition. *Indicates P

Figure 5:. Sub-ablative IR and PARP-inhibition enhance in vivo responsiveness to anti-PD-1 antibodies in MSS and MSI tumors.

(A) Schematic representation of the treatment schedule indicating days animals receive veliparib (25 mg/kg BID, green arrows), radiation (8 Gy, red arrows), or RMP1–14 (100 mg/kg IP, black arrows). Tumor volumes following treatment for CT26 and MC38 tumors are shown in (B-C) and survival curves are shown in (D-E). (F) MC38-bearing mice were treated with IR, Veliparib, and RMP-14 +/− CD8+ T-cell depletion. N=3 (control), 5–11 (experimental). *indicates p