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2020 Deletion of AMPK in Human T Cells Decreases Glycolytic Compensation and Severity of Xenogeneic Gvhd without Impairing GVL Potential

Program: Oral and Poster Abstracts
Session: 701. Experimental Transplantation: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Research, Translational Research
Saturday, December 7, 2024, 5:30 PM-7:30 PM

Lee-Kai Sun, BS1,2, Archana Ramgopal, DO3, Erica Braverman, MD4, Darlene Monlish, Ph.D.4*, Christopher Wittmann4*, Manda Ramsey4*, Richard Caitley5*, William Hawse, Ph.D.5* and Craig Byersdorfer, MD6

1University of Pittsburgh School of Medicine, Pittsburgh
2UPMC, Department of Pediatrics, Division of Blood and Marrow Transplantation and Cellular Therapies, UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA
3Department of Pediatrics, Division of Blood and Marrow Transplantation and Cellular Therapies, UPMC Children’s Hospital of Pittsburgh, Pittsburgh, PA
4UPMC, Department of Pediatrics, Division of Blood and Marrow Transplantation and Cellular Therapies, UPMC Children’s Hospital of Pittsburgh, Pittsburgh, PA
5University of Pittsburgh, Department of Immunology, Pittsburgh, PA
6University of Pittsburgh, Pittsburgh, PA

Background: Allogeneic hematopoietic stem cell transplantation is a curative therapy for hematological disorders but is often limited by graft-versus-host-disease (GVHD). Following transplantation, alloreactive T cells rapidly increase metabolism which presents a promising area for therapeutic intervention. We have previously shown that GVHD-causing murine T cells upregulate signaling through the cellular energy sensor AMP-activated protein kinase (AMPK), and that transfer of AMPK KO T cells increased recipient survival while preserving leukemia clearance. Here, we follow-up these original results with further mechanistic data and translational studies in AMPK-deficient human T cells.

Methods: AMPK-deficient human T cells, generated by targeting the AMPKa1 locus via CRISPR cas9 editing, were transplanted with autologous APCs into immunodeficient (NSG) mice. To test anti-leukemia efficacy, AMPK was deleted in both unmanipulated and CD19-targeting CAR T cells, followed by coculture with either Molm13 or Nalm6 GFP+ leukemia cells in the Incucyte analyzer. Murine T cells lacking AMPK were transplanted into B6D2F1 recipients and proteins immunoprecipitated from day 7 cells using an antibody recognizing the phosphorylated AMPK specific motif (LxRxx(pS/pT)). For inhibitor studies, murine T cells were treated with SBI-0206965 for 24 hours prior to allogeneic transplant.

Results: Transplantation of AMPK-deficient human T cells improved recipient survival including median day of survival (not reached vs. 78 days) and percentage alive at twelve weeks post-transplant (89 vs. 45%, p=0.041). Furthermore, deletion of AMPK lessened GVHD clinical scores as early as day 50, a phenotype which persisted through study end (p<0.0001). A second set of human T cells recovered at 3-4 weeks post-transplant exhibited decreased CD4/CD8 ratios (p<0.05) and impaired glycolytic compensation (p<0.001). This finding is consistent with data from day 7 murine cells showing decreased immunoprecipitation of AMPK-phosphorylated glycolytic enzymes including aldolase and GAPDH (p<0.001 and <0.01, respectively). Importantly, both polyclonal and CD19-directed CAR cytotoxicity was preserved in AMPK-deficient human T cells when cocultured with two separate types of leukemia cells. Finally, pretreatment of murine T cells with the AMPK inhibitor, SBI-0206965, reduced the frequency of proliferating allogeneic T cells recovered on day 7 post-transplant.

Discussion: Here we show for the first time that deletion of AMPK in human T cells minimizes GVHD severity, with a concomitant decrease in donor CD4/CD8 ratios and an impairment in T cell glycolytic compensation. Maintenance of cytotoxicity across multiple in vitro leukemia models further demonstrates the preservation of intrinsic GVL responses in AMPK deficient cells. Importantly, pretreatment of murine T cells with an AMPK inhibitor decreased allogeneic T cell proliferation post-transplant, portending the potential for future clinical translation. Further studies will explore AMPK inhibitor pretreatment of T cells in additional models of murine and human GVHD.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH