Chimeric antigen receptor (CAR) T-cell therapies, while promising for CD19+ hematologic malignancies, often face setbacks due to relapses. Our research identified G protein–coupled receptor 65 (GPR65) as a tumor-specific determinant affecting the efficacy of CAR T-cell therapy. In human patients and an immune-competent mouse model of B-cell acute lymphoblastic leukemia, low GPR65 expression correlates with resistance to CD19+ CAR T treatment. GPR65 knockout (GPR65 KO) tumors in mice similarly exhibit resistance. Through single-cell network analyses, we discovered that GPR65 deficiency reshapes tumor interactions with host macrophages by increasing tumor VEGFA levels, leading to macrophage expansion and preferential M2 polarization. Depleting host macrophages or deleting VEGFA in GPR65 KO tumors restores CAR T-cell therapy responsiveness. Moreover, combining anti-VEGFA therapy with CAR T-cell treatment significantly prolongs the survival of mice bearing GPR65 KO tumors. These findings emphasize the profound impact of tumor gene expression on the tumor microenvironment and subsequent CAR T-cell therapy outcomes.

Significance:

The study identifies GPR65 as an important determinant of B-cell acute lymphoblastic leukemia response to CAR T-cell therapy. Notably, GPR65 absence signals CAR T resistance. By emphasizing the therapeutic potential of targeting VEGFA or host macrophages, our study identifies routes to optimize CAR T-cell therapy outcomes in hematologic malignancies via tumor microenvironment manipulation.

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©2025 The Authors; Published by the American Association for Cancer Research
2025
American Association for Cancer Research
This open access article is distributed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license.
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First page of GPR65 Inactivation in Tumor Cells Drives Antigen-Independent CAR T-cell Resistance via Macrophage Remodeling<alt-title alt-title-type="short">GPR65-Deficient Tumors Are Resistant to CAR T-cell Therapy</alt-title>

Supplementary data

Supplementary Tables S1-S5

Table S1: Differential expression and Activity analysis of human (h.CR vs h.NR) and mouse (m.CR vs m.PR) B-ALL tumor samples. The colum is ranked by combined Z-score of human and mouse DE results. Table S2: Functional enrichment analysis upregulated and downregulated genes in m.CR vs m.PR tumors RNA-seq. Table S3: Differential Expression analysis results of CART T cells treated with No tumor, CR tumors or GPR65 KO tumors. Table S4: Differential Expression analysis of GPR65 KO vs m.CR tumor cells from Day 7 Untreated, Day 11 Untreated and Day 11 CAR-T cell treated tumors. Table S5: NetBID2 Hidden driver analysis of 1035 TF comparing CAR T Treated GPR65 KO and CR tumors.

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Supplementary Figure S1

Figure S1 shows that GPR65 is a biomarker of B-ALL immunotherapy response

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Supplementary Figure S2

Figure S2 shows the strategy to generate GPR65 clones.

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Supplementary Figure S3

Figure S3 shows that GPR65 KO mediated CAR-T resistance is independent of antigen expression and CAR-T cell expansion.

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Supplementary Figure S4

Figure S4 shows CAR-T cell function remains unimpaired in GPR65 KO TME.

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Supplementary Figure S5

Figure S5 is the scRNA-seq experiment design to evaluate tumors and TME.

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Supplementary Figure S6

Figure S6 shows characterization of mouse and human B-ALL TME.

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Supplementary Figure S7

Figure S7 shows macrophage depletion and CAR-T cell therapy regimen.

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Supplementary Figure S8

Figure S8 shows cell-cell communication analysis of tumors, macrophages, and monocytes.

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Supplementary Figure S9

Figure S9 shows that anti-VEGFA sensitizes GPR65 KO tumors to CAR-T cell therapy.

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Supplementary Figure S10

Figure S10 shows that GPR65 KO upregulates VEGFA in tumor cells via FOXO1 network.

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