Abstract 3904: Multiplexed flow cytometry based immunophenotyping paired with functional ex vivo (MFLEX) drug profiling informs potential efficacy of therapeutic agents in acute myeloid leukemia

Abstract

Abstract Introduction: Acute Myeloid Leukemia (AML) is a heterogenous hematological malignancy with a complex clonal architecture that requires in-depth immunophenotyping for disease characterization. Conventional AML ex vivo models for preclinical drug screening lack functional readouts that provide single cell level data to identify drug activity in defined AML subsets with unique immune signatures. Here, we present a novel platform that links Multiparameter Flow cytometry with high-throughput EX vivo drug screening (MFLEX) in AML patient-derived cell models to simultaneously detect phenotypic changes and predict cell-subset specific drug responses in AML. Method: We developed a 21-color high-dimensional immune profiling panel that captures AML heterogeneity by delineating primitive and mature AML blasts along with normal hematopoietic lineages. The panel also includes markers for functional measurement of apoptosis, proliferation, differentiation, and expression of BCL2 family proteins in lymphoid and myeloid cell subsets. Ex vivo culture conditions were optimized using physiologically relevant stroma-free cytokine-rich media that supports primary AML cells with minimal phenotypic shift, allowing for high throughput functional drug sensitivity testing. Timepoints and dosing conditions for AML standard of care (SoC) agents were optimized to support combination screens. Multi-parameter datasets were analysed for functional differences and visualized using dimension reduction methods. Results: We tested MFLEX on clinically annotated AML samples to understand sensitivity profiles to venetoclax and combination partners: 5-azacytidine, cytarabine, fludarabine and gilteritinib. We identified distinct drug sensitivity profiles for venetoclax in AML patients with varying leukemic differentiation states where M0/M1 subtypes were more sensitive to therapy than M4/M5. We also observed cell lineage specific sensitivities, with myeloid blast being more sensitive to venetoclax compared to autologous CD4+ and CD8+ T-cells and this observed difference was correlated with higher expression of BCL2. Our platform demonstrated that patients who previously failed venetoclax therapy could benefit from a combination with 5-azacytidine or fludarabine and this was patient-specific. We further evaluated MFLEX’s predictive value for patient clinical response by systematically comparing venetoclax sensitivity in patient-derived xenograft models in vivo vs ex vivo and found a high correlation between drug responses. Conclusion: We have established a unique, immune drug screening platform, MFLEX, that has high translational value and can be implemented in clinical trials to identify drug response patterns, novel combinations, and potential biomarkers of response for patient stratification in AML. Citation Format: Reecha Shah, Heba Wander, Michelle Hsueh, Muskan Floren, Wei Liu, Patricia Cheung, Courtney Andersen, Michael White, Nathan Kingston, Pablo Morentin Gutierrez, Lisa Drew, Giulia Fabbri, Elena Bibikova, Daniel Auclair. Multiplexed flow cytometry based immunophenotyping paired with functional ex vivo (MFLEX) drug profiling informs potential efficacy of therapeutic agents in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3904.