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637 Cross-Talk between Leukemic Blasts and T Cells Drives Targetable T Cell Dysfunction in the AML Tumor Microenvironment

Program: Oral and Poster Abstracts
Type: Oral
Session: 618. Acute Myeloid Leukemias: Biomarkers and Molecular Marker in Diagnosis and Prognosis: Deciphering the Mechanisms Underlying Prognosis
Hematology Disease Topics & Pathways:
Research, Translational Research, Immune mechanism, Diseases, Immunology, Myeloid Malignancies, Biological Processes
Sunday, December 8, 2024: 4:30 PM

Susan DeWolf, MD1, Nicholas Ceglia, PhD2*, Matthew Zatzman, PhD3*, Brianna Gipson4*, Yuval Elhanati, PhD5*, Zoe Katsamakis6*, Hannah Lees, PhD3*, Kenyon Weis7*, Gustavo Yepes8*, Barbara Oliveira8*, Ana Figueiredo Longhini8*, Natalie Grier Smith, BA9*, Katherine Nichols4*, Eliyahu Havasov3*, Karen Zhao8*, Brigita Meskauskaite8*, Ignas Masilionis8*, Alexander M. Lewis7*, Maria Adriana Cuibus8*, M. Kazim Pajwani, PhD8*, Simone A Minnie, PhD10, Ronan Chaligne, PhD11*, Andri Leo L Lemarquis, MD, PhD4*, Aaron D. Goldberg12, Katharine C. Hsu, MD, PhD4, Eytan M. Stein, MD13, Benjamin D. Greenbaum, PhD3*, Omar Abdel-Wahab, MD1, Sohrab P. Shah, PhD2* and Marcel R.M. van den Brink, MD, PhD14

1Leukemia Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY
2Computational Oncology, Memorial Sloan Kettering Cancer Center, New York, NY
3Computational Oncology, Memorial Sloan Kettering Cancer Center, New York
4Memorial Sloan Kettering Cancer Center, New York, NY
5Computational Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY
6Department of Pediatrics, Immune Discovery and Modeling Service, Memorial Sloan Kettering Cancer Center, New York, NY
7Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY
8Memorial Sloan Kettering Cancer Center, New York
9Department of Immunology, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY
10Translational Sciences and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA
11Single Cell Analytics and Innovation Lab, Memorial Sloan Kettering Cancer Center, New York, NY
12Department of Medicine; Leukemia Service, Memorial Sloan Kettering Cancer Center, New York, NY
13Department of Medicine; Leukemia Service, Memorial Sloan-Kettering Cancer Center, New York, NY
14Department of Hematology and Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte, CA

Allogeneic hematopoetic cell transplantation harnesses donor T cell alloreactivity against leukemic blasts and is curative in a subset of patients with AML. Aside from transplant, however, T cell based immunotherapies have been unsuccessful in AML and in patients with AML there is evidence for impaired endogenous immune responses. Despite these observations, mechanisms underpinning ineffective anti-leukemic T cell immunity are not fully known.

Here we hypothesized that leukemic blasts drive impaired T cell immunity leading to distinct T cell compositions during different disease states. Using multi-modal approaches to study T cell phenotype and T cell receptor (TCR) repertoire across AML disease states we identified dominant clonally expanded terminal effector memory CD45RA+ (TEMRA) CD8 T cells in the marrow of AML patients with active disease along with abundant immunosuppressive CD4 T regulatory cells (Tregs). CD8 TEMRA clones maintain over time in patients with persistent AML and exhibited numerous interactions with malignant blasts suggestive of ongoing immune modulation by antigen producing leukemic cells. A subset of these CD8 effectors (expressing CX3CR1 and other NK-like markers) exert anti-tumor cytotoxic activity ex vivo but are suppressed by interactions with marrow Tregs. Consistent with this, Treg depletion rescued CD8 effector activity and promoted AML eradication.

To study leukemic blasts and T cell immunity in the AML tumor microenvironment (TME), we performed integrative analysis of protein (CITE-seq and 31-color spectral flow), transcript, and TCRs in individual lymphoid and myeloid cells from longitudinal marrows. We applied this to 179 patient samples from 91 subjects (71 AML, 20 controls), sequencing >670K cells total and >190K T cells. We found that patients harbor a highly abundant CD8 TEMRA population at AML diagnosis that persisted over time in patients who did not achieve remission. These cells express clonally expanded TCRs, a subset of which were marked by CX3CR1, TIGIT (but not PD-1 or TIM3) and attributes of cytotoxicity including granzyme B, perforin, and NK-like markers.

To interrogate the function of expanded effector CD8 T cells, we investigated marrow T cells in an unirradiated syngeneic AML mouse model (C1498 cells into C57B6 mice). As AML accumulated in the marrow, TIGIT+ effector CD8s increased in frequency, as in human AML. A subset of these effectors expressed CX3CR1, which we hypothesized might have tumor killing capability given their cytotoxic profile in patient data. Indeed, in vitro functional analysis revealed increased killing of endogenous tumor by marrow CD8 effectors including CX3CR1+ effectors compared to naïve CD8s harvested 18-20 days post-tumor injection. Importantly in patients with AML we harnessed the TCR CDR3 sequence as a barcode to track phenotypes of CD8 clonotypes over time and found that certain CX3CR1+ TEMRAs transition to a CX3CR1- state in ongoing disease, suggesting loss of cytotoxicity.

Analyses of cell-cell interactions from CITE-seq suggested altered myeloid-T cell interactions in AML compared to control and remission samples, including increased signaling between myeloid cells, Tregs, and memory CD8s in patients with active AML. AML blasts also had increased expression of T cell inhibitory molecules including TIGIT ligands, CD244, and VISTA compared to healthy myeloid cells. Notably, in addition to expanded CD8 TEMRAs, we found increased Tregs in marrow from patients with AML and C1498 engrafted mice. Tregs in both human and mouse AML expressed high levels of TIGIT, CD39, ICOS, and CCR4 but were not clonally expanded. Ex vivo AML marrow Tregs suppressed CD8 effector function. Importantly depletion of Tregs in vivo through transgenic FoxP3 diphtheria toxin receptor mice prolonged host survival, promoted tumor clearance, and led to an increase in marrow CX3CR1+ effector CD8 T cells.

These data demonstrate the active immunologic landscape of the AML bone marrow in both patient samples and mouse models of the disease. We find that although CD8 T cells have the potential for anti-leukemic immunity, their efficacy is impaired by leukemic blasts and suppressive Tregs. These studies suggest Treg-targeting interventions as a therapeutic avenue to overcome the immunosuppressive TME in AML and nominate a host of potentially targetable T cell and blast cell surface proteins that restrain T cell anti-tumor immunity in AML.

Disclosures: Goldberg: AROG: Research Funding; Pfizer: Research Funding; Aptose: Research Funding; Syndax Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celularity: Research Funding; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; DAVA Oncology: Honoraria; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ikena Oncology: Consultancy; Kura Oncology: Honoraria, Research Funding; Molecular Partners: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Aprea: Research Funding. Hsu: Exelixis: Consultancy; Wugen, Inc: Membership on an entity's Board of Directors or advisory committees. Stein: Genentech: Consultancy, Other: consulting fees; Jazz Pharmaceuticals: Consultancy, Other: consulting fees; Gilead: Consultancy, Other: consulting fees; Agios Pharmaceuticals: Consultancy, Other: consulting fees; Astellas Pharmaceuticals: Consultancy, Other: consulting fees; Servier: Consultancy, Other: consulting fees; AstraZeneca: Consultancy, Other: consulting fees; Abbvie: Consultancy, Other: consulting fees; Daiichi Sankyo, Inc.: Consultancy, Other: consulting fees; Celgene: Consultancy, Other: consulting fees. Greenbaum: Rome Therapeutics: Consultancy, Other: Co-founder; Shennon Biotechnologies: Consultancy; PMV Pharma: Consultancy; Darwin Health: Consultancy; Chugai Pharmaceuticals: Honoraria; Bristol Meyers Squibb: Honoraria, Research Funding; Merck: Consultancy, Honoraria, Research Funding. Abdel-Wahab: Minovia Therapeutics: Consultancy, Research Funding; Nurix Therapeutics: Research Funding; Codify Therapeutics: Consultancy, Current equity holder in private company, Research Funding. Shah: AstraZeneca Inc: Consultancy; Bristol Myers Squibb: Research Funding.

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