Epigenetic priming of Bladder cancer cells improves CAR T-cell cytotoxicity

Key Points Dasatinib potently and reversibly suppresses CAR-T cell cytotoxicity, cytokine secretion, and proliferation. Dasatinib could be repurposed as a safety switch to mitigate CAR-mediated toxicity in patients.

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Integrated drug profiling and CRISPR screening identify essential pathways for CAR T-cell cytotoxicity

Blood ◽

10.1182/blood.2019002121 ◽

2020 ◽

Vol 135 (9) ◽

pp. 597-609 ◽

Cited By ~ 17

Author(s):

Olli Dufva ◽

Jan Koski ◽

Pilvi Maliniemi ◽

Aleksandr Ianevski ◽

Jay Klievink ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Death Receptor ◽

Cell Cytotoxicity ◽

Car T Cell ◽

Small Molecule Drugs ◽

Car T ◽

Smac Mimetics ◽

Death Receptor Signaling ◽

T Cell Cytotoxicity

Abstract Chimeric antigen receptor (CAR) T-cell therapy has proven effective in relapsed and refractory B-cell malignancies, but resistance and relapses still occur. Better understanding of mechanisms influencing CAR T-cell cytotoxicity and the potential for modulation using small-molecule drugs could improve current immunotherapies. Here, we systematically investigated druggable mechanisms of CAR T-cell cytotoxicity using >500 small-molecule drugs and genome-scale CRISPR-Cas9 loss-of-function screens. We identified several tyrosine kinase inhibitors that inhibit CAR T-cell cytotoxicity by impairing T-cell signaling transcriptional activity. In contrast, the apoptotic modulator drugs SMAC mimetics sensitized B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma cells to anti-CD19 CAR T cells. CRISPR screens identified death receptor signaling through FADD and TNFRSF10B (TRAIL-R2) as a key mediator of CAR T-cell cytotoxicity and elucidated the RIPK1-dependent mechanism of sensitization by SMAC mimetics. Death receptor expression varied across genetic subtypes of B-cell malignancies, suggesting a link between mechanisms of CAR T-cell cytotoxicity and cancer genetics. These results implicate death receptor signaling as an important mediator of cancer cell sensitivity to CAR T-cell cytotoxicity, with potential for pharmacological targeting to enhance cancer immunotherapy. The screening data provide a resource of immunomodulatory properties of cancer drugs and genetic mechanisms influencing CAR T-cell cytotoxicity.

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Epigenetic Priming of Bladder Cancer Cells With Decitabine Increases Cytotoxicity of Human EGFR and CD44v6 CAR Engineered T-Cells

Frontiers in Immunology ◽

10.3389/fimmu.2021.782448 ◽

2021 ◽

Vol 12 ◽

Author(s):

Camilla M. Grunewald ◽

Corinna Haist ◽

Carolin König ◽

Patrick Petzsch ◽

Arthur Bister ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

Antigen Expression ◽

Cell Cytotoxicity ◽

Malignant Cells ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

T Cell Cytotoxicity

BackgroundTreatment of B-cell malignancies with CD19-directed chimeric antigen receptor (CAR) T-cells marked a new era in immunotherapy, which yet has to be successfully adopted to solid cancers. Epigenetic inhibitors of DNA methyltransferases (DNMTi) and histone deacetylases (HDACi) can induce broad changes in gene expression of malignant cells, thus making these inhibitors interesting combination partners for immunotherapeutic approaches.MethodsUrothelial carcinoma cell lines (UCC) and benign uroepithelial HBLAK cells pretreated with the DNMTi decitabine or the HDACi romidepsin were co-incubated with CAR T-cells directed against EGFR or CD44v6, and subsequent cytotoxicity assays were performed. Effects on T-cell cytotoxicity and surface antigen expression on UCC were determined by flow cytometry. We also performed next-generation mRNA sequencing of inhibitor-treated UCC and siRNA-mediated knockdown of potential regulators of CAR T-cell killing.ResultsExposure to decitabine but not romidepsin enhanced CAR T-cell cytotoxicity towards all UCC lines, but not towards the benign HBLAK cells. Increased killing could neither be attributed to enhanced target antigen expression (EGFR and CD44v6) nor fully explained by changes in the T-cell ligands PD-L1, PD-L2, ICAM-1, or CD95. Instead, gene expression analysis suggested that regulators of cell survival and apoptosis were differentially induced by the treatment. Decitabine altered the balance between survival and apoptosis factors towards an apoptosis-sensitive state associated with increased CAR T-cell killing, while romidepsin, at least partially, tilted this balance in the opposite direction. Knockdown experiments with siRNA in UCC confirmed BID and BCL2L1/BCLX as two key factors for the altered susceptibility of the UCC.ConclusionOur data suggest that the combination of decitabine with CAR T-cell therapy is an attractive novel therapeutic approach to enhance tumor-specific killing of bladder cancer. Since BID and BCL2L1 are essential determinants for the susceptibility of a wide variety of malignant cells, their targeting might be additionally suitable for combination with immunotherapies, e.g., CAR T-cells or checkpoint inhibitors in other malignancies.

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Abstract 2790: The monocyte lineage is required for production of clinically relevant interleukin-6 (IL-6) during chimeric antigen receptor (CAR) T cell cytotoxicity

10.1158/1538-7445.am2014-2790 ◽

2014 ◽

Author(s):

David M. Barrett ◽

Nathan Singh ◽

Bruce Levine ◽

Stephan A. Grupp

Keyword(s):

T Cell ◽

Chimeric Antigen Receptor ◽

Interleukin 6 ◽

Antigen Receptor ◽

Cell Cytotoxicity ◽

Car T Cell ◽

Car T ◽

T Cell Cytotoxicity

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Effect of dendritic cells (DC) transduced with chimeric antigen receptor (CAR) on CAR T-cell cytotoxicity.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.7_suppl.144 ◽

2017 ◽

Vol 35 (7_suppl) ◽

pp. 144-144 ◽

Cited By ~ 3

Author(s):

Hyung Chan Suh ◽

Katherine Pohl ◽

Anna Patricia L. Javier ◽

Dennis J. Slamon ◽

John P Chute

Keyword(s):

T Cells ◽

T Cell ◽

Critical Role ◽

Cell Cytotoxicity ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Mock Control ◽

T Cell Cytotoxicity

144 Background: T cells interacting DC could be superior in T cell cytotoxicity. CD141+/Cleg9a+ intra-tumoral DC play a critical role in tumor cytotoxicity. Therefore, combining intra-tumoral DC in CAR T cell would safely increase localized CAR T cell cytotoxicity. We hypothesized that bioengineered DC compartment could be an excellent source for enhanced CAR T cell cytotoxicity. Methods: DC precursors and T cells of PBMC were transduced with a CAR (pCCL-anti-CD33-4-1BB-CD3z-T2A-GFP; CAR-DC or CAR T). For comparison, additional DC were transduced with 4-1BB cDNA (pCCL-4-1BB-T2A-GFP; 4-1BB-DC) or mock control (pCCL-eGFP). In addition to lentivirus transduction, differentiation of DC in vitro employed Flt3L/GM-CSF/IL-4. Transduced CAR T and CAR-DC were sorted by GFP expression at day 5. After further 10 days of culture, cells were harvested and analyzed for phenotype. An acute myeloid leukemia (AML) cell line (Kasumi-1) was treated with CAR T +/- CAR-DC, 4-1BB-DC, or mock control for functional assays. Results: Frequencies of cells expressing CD141+/Cleg9a+ were higher in 4-1BB-DC vs. control DC (33% vs. 1.5%). After mixing CAR T and CAR-DC (5X105) with Kasumi-1 (1X105) for 6 hours, CAR T/CAR-DC showed 100% Kasumi-1 cell cytotoxicity compared to 70% of CAR T by trypan blue. CAR T/CAR-DC also demonstrated higher Annexin V positive Kasumi-1 cells compared with CAR-T (91% vs. 52%). CAR T with or without CAR-DC were also assessed with multiplex immunoassays. CAR T cells mixed with CAR-DC induced higher level of IFN-gamma (10,316 vs. 6,186 pg/ml), IL-2 (68,840 vs. 64,708 pg/ml), and TNFalpha (1,361 vs. 905 pg/ml) (Kasumi-1 cells mixed with CAR-T cells of 10 E/T ratio) than CAR T cells. CAR-DC produced significantly higher IL-12 cytokine production (1,352 vs. 161 pg/ml) than CAR T cells in response to CD33 but independent to T cells, confirmed by comparing IL-12 production with CAR T/4-1BB-DC. Conclusions: These data show that in vitro differentiation of DC bearing 4-1BB increases CD141+/Cleg9a+ DC population and that interaction with CAR-DC to CAR T cells enhances anti-AML cytotoxicity. Our finding may implicate the development of CAR-DC therapy combined to CAR T cells to increase the efficiency of cancer immunotherapy.

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