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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.

Tumor cells upregulate myriad proteins that are important for pH regulation, resulting in the acidification of the extracellular tumor microenvironment (TME). Abnormal pH is known to dampen immune function, resulting in a worsened anti-tumor immune response. Understanding how extrinsic alterations in pH modulate the interactions between immune cells and tumors cells will help elucidate opportunities for new therapeutic approaches. We observed that pH impacts the function of immune cells, both natural killer (NK) and T cells, which is relevant in the context of a highly acidic TME. Decreased NK and T cell activity was correlated with decreasing pH in a co-culture immune cell-mediated tumor cell-killing assay. The addition of pH-modulating drugs cariporide, lansoprazole, and acetazolamide to the co-culture assay was able to partially mitigate this dampened immune cell function. Treatment of colorectal cancer (CRC) cells with NHE1 inhibitor cariporide increased CRC cell-secreted cytokines involved in immune cell recruitment and activation and decreased cytokines involved in epithelial-mesenchymal transition (EMT). Cariporide treatment also decreased CRC cell shed TRAIL-R2, TRAIL-R3, and PD-L1 which is relevant in the context of immunotherapy. These experiments can help inform future investigations into how the pH of the tumor microenvironment may be extrinsically modulated to improve anti-tumor immune response in solid tumors such as colorectal cancer.

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.

Inflammatory cytokines, immune stimulants, chemokines, and tumor growth/growth suppressing factors are molecular messengers that circulate and have the capability to modify the tumor microenvironment and impact response to therapeutics. The characterization of soluble mediators as biomarkers for diagnosis and prognosis is of interest in oncology. We utilize the cytokinome to characterize the response of colorectal tumor cell lines to selected small-molecules in oncology as a proof-of-concept dataset with immune synergy heat map rankings of analytes for drug and cell line combinations. We observed overall trends in drug-class effects with MEK-, BRAF-, PARP-inhibitors, and Imipridones in cytokine, chemokine, and growth factor responses that may help guide therapy selection. MEK-inhibitor treatment downregulated analytes VEGF, CXCL9/MIG, and IL-8/CXCL8 and upregulated CXCL14/BRAK, Prolactin, and CCL5/RANTES. BRAF-inhibitor treatment downregulated VEGF and IL-8/CXCL8, while increasing sTRAIL-R2. Treatment with PARP-inhibitors decreased CXCL9/MIG, IL-8/CXCL8, CCL3/MIP-1 alpha, VEGF, and CXCL14/BRAK, while treatment increased sTRAIL-R2 and prolactin. Treatment with Imipridones decreased CCL3/MIP-1 alpha, VEGF, CXCL14/BRAK, IL-8/CXCL8, and Prolactin and increased CXCL5/ENA-78. We also observed differential responses to therapeutics depending on the mutational profile of the cell line. In the future, a similar but larger data-set may be utilized in the clinic to aid in immune synergy prediction of a patient's response to therapy based on tumor genotype. Citation Format: Kelsey E. Huntington, Anna Louie, Lanlan Zhou, Wafik S. El-Deiry. A high-throughput customized cytokinome screen of colon cancer cell responses to small-molecule oncology drugs [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P036.