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4803 Reprogramming CLL T-Cell Mitochondrial Fitness Using PI3K Inhibition for Enhancing CAR T-Cell Therapy

Program: Oral and Poster Abstracts
Session: 702. CAR-T Cell Therapies: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Fundamental Science, Research, Translational Research, Immunology, Metabolism, Biological Processes, Molecular biology
Monday, December 9, 2024, 6:00 PM-8:00 PM

Wael Gamal, PhD1,2, Nienke B. Goedhart, MSc3,4,5*, Helga Simon-Molas, PhD3,4,5*, Melanie Mediavilla Varela, PhD2*, Angimar Uriepero Palma, MD, MMSc6*, Fleur S. Peters, PhD3,4,5*, Eva Sahakian, PhD2,7*, Arnon P. Kater, MD, PhD4,5 and Javier Pinilla-Ibarz, MD, PhD7

1Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL
2Department of Immunology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL
3Department of Experimental Immunology, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands
4Cancer Immunology, Cancer Center Amsterdam, Amsterdam, Netherlands
5Department of Hematology, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands
6Department of Internal Medicine, Jefferson Einstein Hospital, Philadelphia, PA
7Department of Malignant Hematology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL

Chronic lymphocytic leukemia (CLL) presents a significant clinical shortfall, as the number of patients developing resistance to both BTK inhibitors and Bcl2-targeted therapies is rapidly increasing. To date, chimeric antigen receptor (CAR) T cells have shown restricted success in CLL, attributed to CLL-induced T-cell dysfunction and a shift toward terminally differentiated cell subsets. The mechanisms behind these T-cell changes are still not well-understood. T-cell activation and differentiation are metabolically demanding processes. Previously, we identified abnormal T-cell metabolic properties in CLL (van Bruggen et al, Blood 2019). The link between metabolic activity and T-cell differentiation indicates that modulating metabolic pathways could improve the effectiveness of T-cell therapies. However, this approach has not yet been explored in CAR T-cell production for CLL. We aim to uncover the relationship between differentiation and metabolism in CLL T cells and identify the factors driving T-cell dysfunction to revitalize CAR T cells.

Flow cytometry, mitochondrial stress tests and metabolomics were used to analyze healthy and CLL patients' T cells at baseline and post-T-cell receptor (TCR) activation. For murine experiments, splenocytes obtained from transgenic Eμ-TCL1 mice were adoptively transferred (AT) into wild-type C57BL/6 mice, and splenic T cells were studied at various disease stages. Regarding ex vivo reprogramming experiments, human or mouse T-cell cultures were treated with the PI3Kδ inhibitor idelalisib. Generation of CAR T cells was done using T cells from AT Eμ-TCL1 mice, stimulated ex vivo with or without idelalisib, and virally transduced with a GFP-tagged m1928z CAR construct. Leukemic mice infused with these CAR T cells were monitored over time.

Mitochondrial depolarization, indicated by a reduced mitochondrial membrane potential relative to mass, was observed in human and murine CLL T cells. T cells from AT Eμ-TCL1 mice demonstrated disturbed mitochondrial function alongside a shift toward terminally differentiated exhausted-like cells during CLL progression. Furthermore, extracellular flux analysis following TCR stimulation indicated diminished mitochondrial spare respiratory capacity in CLL T cells, consistent in both human and mouse models. Metabolomics and 13C fractional labeling demonstrated decreased mitochondrial fueling and lower levels of mitochondrial metabolites in CLL T cells. These observations of metabolic disturbance aligned with an exhausted-like T-cell differentiation phenotype controlled at the epigenetic level. An increase in PI3K/Akt signaling was identified in T cells from AT Eμ-TCL1 mice. Ex vivo reprogramming of human and murine CLL T cells with the PI3Kδ inhibitor idelalisib resulted in metabolically reprogrammed T cells with improved memory formation and mitochondrial activity. Adding idelalisib during the generation of murine CD19 CAR T cells significantly enhanced the in vivo persistence of infused cells in the immunocompetent Eμ-TCL1 mice and led to long-term leukemia-free remissions, underscoring the clinical relevance of these findings.

Overall, we found that the buildup of dysfunctional, depolarized mitochondria is a marker of T-cell metabolic abnormalities associated with terminal differentiation in CLL progression. PI3K inhibition is a potential strategy to improve CLL T-cell metabolic plasticity and enhance the efficacy and persistence of autologous CAR T-cell infusion products.

WG, NG, and HSM share the first co-authorship.

JPI and APK share senior authorship.

Disclosures: Kater: Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; LAVA: Membership on an entity's Board of Directors or advisory committees, Other: Patents planned, issued or pending; Steering Committee; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Patents planned, issued or pending; Steering Committee, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Steering Committee, Research Funding. Pinilla-Ibarz: Bristol Meyers Squibb: Consultancy, Speakers Bureau; Novartis: Honoraria; AbbVie: Consultancy, Speakers Bureau; AstraZeneca: Consultancy, Speakers Bureau; Eli Lily: Consultancy, Speakers Bureau; Secura Bio: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Pfizer: Consultancy; Beigene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau.

*signifies non-member of ASH