LAUSR

Background: The recent discovery that multiple chemo- and immuno-therapies activate non-apoptotic cell death pathways such as necroptosis, pyroptosis, and ferroptosis in cancer cells has necessitated development of pharmacodynamic biomarker assays to study their novel mechanisms of action. We describe the development, analytical validation, and proof-of-concept demonstration of assays for potential biomarkers of ferroptosis, pyroptosis and necroptosis pathways to support anti-cancer drug development. Methods: We developed multiplex sandwich immunoassays on the Luminex platform to quantify transferrin receptor (TfR), glutathione peroxidase 4 (GPx4), active caspase-3, cleaved and full-length gasdermins D and E (GSDMD, GSDME), MLKL oligomers, and MLKL heterodimers with RIPK1 and RIPK3. Novel antibodies to gasdermin cleavage neoepitopes were generated and their specificity validated with knockout (GSDMD) or non-expressing (GSDME) cell lines. We utilized well-established in vitro cancer cell line models (NCI-H522, THP-1, MV411, MB231, HT29) and inducers/inhibitors of pyroptosis, necroptosis and ferroptosis to test for expected directional changes of biomarker modulation. Results: Analytical validity of the measurements is demonstrated with acceptable reproducibility, precision, dilutional linearity, dilutional recovery, spike recovery, sample freeze-thaw and temperature stability. The fitness-for-purpose of the assays is demonstrated by increased cleavage of GSDME, loss of full-length GSDME, and activation of caspase-3 in NCI-H522 cells treated with doxorubicin; similarly, cleavage of GSDMD and loss of full-length GSDMD is shown in THP-1 cells primed with LPS and treated with nigericin, and in MV-4-11 cells treated with val-boroPro. HT29 cells treated with ZVAD + birinapant + TNFα were used to demonstrate increased MLKL oligomerization or heterodimer formation with RIPK1/RIPK3. Modulation of ferroptosis biomarkers TfR and GPx4 (SU-DHL-5, MDA-MB-231) in response to imidazole ketone erastin (IKE) or RSL3 was cell line-dependent. Studies are underway to validate modulation of select ferroptosis biomarkers using orthogonal LC-MS methods. Conclusions: We describe development and translational readiness of novel multiplex assays to interrogate drugs inducing ferroptosis, pyroptosis and necroptosis cell death pathways. Our data demonstrate utility of biomarkers of pyroptosis and necroptosis, whereas biomarkers of ferroptosis require further appraisal. In vivo pharmacodynamic studies of pyroptosis, ferroptosis and necroptosis inducers are ongoing to verify assay fitness-for-purpose with tumor biopsy specimens. This project was funded with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: William G. Herrick, Jeevan Govindharajulu, Ralph E. Parchment, James H. Doroshow, Apurva K. Srivastava. Novel multiplexed assays of ferroptosis, pyroptosis and necroptosis biomarkers for translational studies [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 6143.