(M1430-03-17) Novel Synergistic TKI Combination Induces Immunogenic Cell Death in Colon Cancer with BRAF and KRAS Mutations

Purpose: Tyrosine kinase inhibitors (TKIs) are small molecules that selectively or broadly inhibit tyrosine kinases, key regulators of signaling pathways involved in cancer cell proliferation, survival, and metastasis. Since their clinical introduction in 2001, TKIs have been approved as first- and second-line therapies for various malignancies, including chronic myeloid leukemia, non-small cell lung cancer (NSCLC), thyroid cancer, and melanoma. Despite the availability of numerous TKIs, only a limited subset such as regorafenib, sotorasib, adagrasib, and tucatinib have received approval for colorectal cancer treatment, primarily due to issues of acquired resistance and systemic toxicity. To address these limitations, this study focuses on identifying a synergistic TKI combination capable of inducing immunogenic cell death (ICD) and enhancing therapeutic efficacy.

Methods: The drug screening process was examined in this study in two distinct stages. First, the cytotoxicity of drugs from various classes was examined, and synergistic drug combinations were identified using the Bliss assay model. Second, the selected drug combinations were evaluated in the HT-29 xenograft and a CT-26 syngeneic mouse model. All the drugs were delivered at the same route of administration and dosing frequency, as conducted in the clinic. To check the immunogenicity of synergistic trametinib and nintedanib combination, ATP¹ and calreticulin assay² were performed.

Results: Analysis of Bliss assays demonstrated that trametinib acted in synergy with most of the drugs tested (Figure 1a). Combining trametinib with one of five selected drugs and comparing these five combinations in a therapeutic HT-29 (BRAF mutant) xenograft model showed that the trametinib and nintedanib (TN) combination had greater tumor regression compared to all other tested groups (Figure 1b). The same group when tested in CT26 (KRAS mutant) syngeneic model, slowed down the tumor growth during treatment period and combination with immune checkpoint blockade (anti-PD-1) further delayed the tumor growth and enhanced the overall survival of mice (Figures 1c and 1d). To understand the immunogenicity of the combination ATP and calreticulin assay were performed. Figure 2a demonstrates that ATP release was significant during TN combination treatment rather than single therapy. Similarly, calreticulin release was strongly associated with combination treatment (Figure 2b).

Conclusion: From the in vitro and in vivo drug screening studies, we conclude that trametinib and nintedanib combination is promising for both BRAF and KRAS mutated colon cancer models. Additionally, this combination induces immunogenic cell death, which could be a promising approach for colon cancer.

References: 1. Zhang, J., Sun, X., Zhao, X., Yang, C., Shi, M., Zhang, B., Hu, H., Qiao, M., Chen, D., & Zhao, X. (2022). Combining immune checkpoint blockade with ATP-based immunogenic cell death amplifier for cancer chemo-immunotherapy. Acta Pharmaceutica Sinica B, 12(9), 3694–3709. https://doi.org/10.1016/j.apsb.2022.05.008
2. Tesniere, A., Schlemmer, F., Boige, V. et al. Immunogenic death of colon cancer cells treated with oxaliplatin. Oncogene 29, 482–491 (2010). https://doi.org/10.1038/onc.2009.356

Acknowledgements: This work was financially supported by Gastro-Intestinal Research Foundation (GIRF)

Synergistic combination screening studies. a) synergy scores of different drug combinations obtained from bliss assay model (orange-red: synergism, yellow: additive and green: antagonism), tumor regression studies in b) HT-29 xenograft model, c) CT 26 syngeneic model and d) Kaplan-Meier survival plot of CT-26 model mice.

Immunogenic cell death assessment. a) ATP assay and b) Calreticulin assay (x axis indicates florescence signals of calreticulin and Y axis represents propidium iodide, PI)