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Supplementary Methods

Supplementary Table S1: list of antibodies used in this study for western blots (WB), immunofluorescence (IF) and immunohistochemistry (IHC.)

Supplementary Table S2: differential blood cell count of mice treated with ONC212, 2-DG, or the combination of both agents.

Supplementary Table S3: blood chemistry results of mice treated with ONC212 or the combination of ONC212 and 2-DG

Thoracic therapeutic ionizing radiation is limited by toxicities such as pneumonitis and fibrosis of the lungs. Such limitation restricts therapeutic doses and adversely affects patient quality of life while undergoing and following treatment. ONC201/TIC10 is a small-molecule anti-cancer drug that activates the integrated stress response (ISR) and drives the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway. Pegylated recombinant long-acting TRAIL (TLY012) has been shown in preclinical models to induce the reversal of fibrosis and currently has orphan drug status for systemic sclerosis and chronic pancreatitis. We show a similar effect of both TLY012 and ONC201 in vivo in protecting from radiation pneumonitis and fibrosis of the lungs. WT and TRAIL-/- C57Bl/6 mice receiving a single 20 Gy thoracic radiation dose with shielding of other organs and treated with 10 mg/kg of TLY012 twice a week showed a significantly reduced alveolar wall thickness and lessened inflammation compared to controls and DR5-/- mice receiving the same treatment upon histological analysis of the lungs conducted 13 days post-irradiation. WT and TRAIL-/- C57Bl/6 mice treated with 100 mg/kg of ONC201 once a week showed similar effect to a lesser extent. Further analysis in C57Bl/6 WT mice bearing orthotopic mammary fat pad e0771 TNBC tumors similarly receiving a single 20 Gy thoracic radiation dose revealed the same pattern of protection from radiation pneumonitis upon TRAIL-pathway agonism through treatment with TLY012 and ONC201 both in combination and alone, while also showing a significant reduction in tumor burden at the experimental endpoint (day 9 post-irradiation) in the combination treated mice. Further, pulse oximetry readings of the hind paw revealed a notable reduction in oxygen saturation in all mice except those treated with TLY012. Cytokinomic profiling of mouse serum upon sacrifice revealed a significant reduction in CCL22/MDC levels in the TLY012 cohort. Additional post-hoc analysis including immunophenotyping, immunostaining, and bulk RNA analysis through Nanostring nCounter technologies is underway. Altogether, these findings suggest a role for TLY012, ONC201, or broader modulation of the TRAIL/DR5 pathway in mitigating adverse effects and outcomes of therapeutic radiation, and may serve as a foundation for safer use of radiation in the clinic. Citation Format: Jillian R. Strandberg, Anna Louie, Marina Hahn, Praveen Srinivasan, Andrew George, Arielle De La Cruz, Leiqing Zhang, Liz Hernandez Borrero, Kelsey Huntington, Christopher Azzoli, Abbas E. Abbas, Lanlan Zhou, Seulki Lee, Wafik S. El-Deiry. Post exposure suppression of radiation pneumonitis by TRAIL pathway agonists TLY012 and ONC201. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4457.

Immune checkpoint blockade (ICB) with αPD-1/PD-L1 has impressive efficacy in microsatellite unstable (MSI+) CRC but no significant activity in the 85% of CRC cases that are microsatellite stable (MSS). Our group previously demonstrated that 9-ING-41, a small-molecule inhibitor of GSK-3 in clinical development, stimulates both NK and T cell activity. This project characterizes the effects of 9-ING-41 in combination with ICB in vivo. In a syngeneic murine colon carcinoma BALB/c model using MSS cell line CT-26, we compared isotype, 9-ING-41 (70 mg/kg 2x/wk), αPD-1 (10 mg/kg 2x/wk), αPD-L1 (10 mg/kg 2x/wk), 9-ING-41 + αPD-1, and 9-ING-41 + αPD-L1 treatment groups. The median overall survival was 19 days (d) with 9-ING-41 (hazard ratio for death (HR) 0.12; 95% confidence interval (CI), 0.03-0.54; p=0.008), 16 d for 9-ING-41 + isotype (HR 0.40; 95% CI, 0.08-1.9; p=0.14), 20.5 d for αPD-1 (HR 0.15; 95% CI, 0.03-0.67; p=0.0185), 18 d for αPD-L1 (HR 0.21; 95% CI, 0.05-0.89; p=0.0559), 17 d for 9-ING-41 + αPD-1 (HR 0.37; 95% CI, 0.09-0.1.5; p=0.29), and 45.5 d for 9-ING-41 + αPD-L1 (HR 0.08; 95% CI, 0.02-0.36; p=0.0019), compared to 16 d for the isotype control group. Tumor response rates observed were 33.3% in the 9-ING-41 + αPD-L1 group, 16.6% in the αPD-1 group, and a 0% for all other treatment groups. We hypothesize that the 9-ING-41-mediated upregulation of PD-L1 in CRC cells (HCT-116, HT-29) observed via both flow cytometry (FC) and western blot analysis may contribute to the increased efficacy of combination therapy with αPD-L1, as compared to αPD-1. Significant differences between responders and non-responders in intratumoral and splenic natural killer (NK) and T cell subsets 14-days post-treatment initiation were shown by multi-color FC. Compared to non-responders, regardless of treatment group, responders had lower percentages of splenic CD4+ (p=0.0145) and CD8+ T cells (p=0.0001), increased percentages of splenic CD69+ activated T cells (p=0.0070) and FOXP3+ regulatory T cells (p=0.001), and increased percentages of tumor-infiltrating CD3+ (p=0.0006) and CD4+ T cells (p Citation Format: Kelsey E. Huntington, Anna Louie, Lanlan Zhou, Benedito A. Carneiro, Wafik El-Deiry. Small-molecule inhibition of glycogen synthase kinase-3 (GSK-3) increases the efficacy of anti-PD-L1 therapy in a murine model of microsatellite stable colorectal cancer (CRC); Therapeutic response correlates with T cell ratios and serum cytokine profiles in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4166.