Fig 2: CD84 loss impairs AML development in PDX.(A) Schematic of the design and procedures of generating a CD84 knockdown, AML PDX model. AML primary patient cells were transduced with shCtrl or shCD84 lentivirus. After puromycin selection, shCtrl or shCD84 AML primary cells were injected into irradiated NSG mice. (B) Representative flow cytometry profile of human AML cells (human CD45+/CD33+) engrafted in BM. (C–E) Scatter plots showing the percentage of human AML cells (human CD45+/CD33+) engrafted in BM (C), SP (D), and PB (E) of recipient NSG mice (n = 5 per group). Data are represented as mean ± SEM and are representative of 5 individual mice per group. Statistical significance was assessed by 2-tailed unpaired t test. (F) Bioluminescent imaging showing the tumor burden in xenograft NSG mice (frontal and dorsal) following shCtrl- or shCD84-transduced AML PDX-luciferase cell transplantation (n = 4 per group). (G) Kaplan-Meier survival analysis of AML PDX-luciferase cell–transplanted (shCtrl or shCD84) NSG mice (n = 4 per group). Statistical significance was assessed by log-rank test. (H) Flow cytometry profile showing apoptosis levels indicated by annexin-APC/DAPI in 32D cells transfected with lentivirus including CD823-mock vector or CD823-CD84 WT. (I) Violin plot showing apoptosis levels indicated by annexin V-APC/DAPI in 32D cells transduced with mock or CD84 WT. Data are represented as mean ± SEM and are representative of 3 biological replicates. Statistical significance was assessed by 2-tailed unpaired t test.

Fig 3: CD84 deletion dampens AML survival in both AML cell lines and cell-derived xenograft.(A) Western blot of the indicated proteins in THP1 cells and HEL cells transduced with 2 shRNAs against CD84 (shCD84-1; shCD84-2) or scramble control (shCtrl). Data are representative of at least 2 independent experiments. (B) Cell proliferative analysis of THP1 cells and HEL cells transduced with shCtrl or shCD84 lentiviral vectors. (C) Bar chart showing apoptosis levels indicated by annexin-APC/DAPI in 3 AML patient specimens transduced with shCtrl or shCD84 lentiviral vector. (D and E) AML cells obtained from 3 different donors (AML #1, #3, #8) were transduced with shCtrl or shCD84 lentivirus. Representative colony images are in D. The graph in E shows AML colony-formation cell (CFC) frequencies after 10 days of culture. n = 3 independent replicates per sample. (F) Bioluminescent imaging showing the tumor burden in xenograft NSG mice on days 14–35 following shCtrl- or shCD84-transduced THP1-luciferase cell transplantation (n = 5 per group). (G) Kaplan-Meier analysis of survival of THP1-luciferase cell–transplanted (shCtrl or shCD84) NSG mice. Each group consisted of 5 mice. (H) Bioluminescent imaging showing the tumor burden in xenograft NSG mice on days 19–41 following mock/shCtrl-, CD84-OE/shCtrl–, or CD84-OE/shCD84–transduced THP1-luciferase cell transplantation (n = 4 per group). (I) Kaplan-Meier analysis of survival of THP1-luciferase cell–transplanted (mock/shCtrl, CD84-OE/shCtrl, or CD84-OE/shCD84) NSG mice (n = 4 per group). (J) Bar chart showing the CD84 surface expression in BM cells from NSG mice xenografted with THP1 luciferase cells transduced with mock/shCtrl or CD84-OE/shCD84. Data are represented as mean ± SEM and are representative of 3 biological replicates (B and E) and 3 independent experiments (C). Each dot in J represents 1 mouse. Statistical significance was assessed by 2-way ANOVA (B, D, and E); 2-way ANOVA (mix model; C); log-rank test (G and I); and 2-tailed unpaired t test (J).

Fig 4: Knockdown of CD84 triggers mitochondrial stress in AML cells.(A) Representative transmission electron microscopy images of mitochondrial cristae in HEL cells transfected with shCtrl or shCD84 lentivirus. Scale bars: 1 μm (top); 0.5 μm (bottom). The analysis was conducted in at least 2 independent experimental sets. (B) Western blot of indicated proteins was performed in HEL cells transduced with lentivirus expressing either shCtrl or shCD84, indicating mitochondrial dysfunction in CD84 knockdown cells. Data are representative of 2 independent biological replicates. (C) Interleaved scatter plot showing the MMPs that were measured using JC-1 dye for flow cytometry. Data are represented as mean ± SEM and are representative of 3 independent experiments. Statistical significance was assessed by 2-tailed unpaired t test. (D) Representative flow cytometry profiles of JC-1–stained THP1, which was transduced with lentiviruses expressing indicated vectors (shCD84 targeting 3′-UTR). Red (PE) and green (FITC) represent the monomers to aggregated ratio. (E) Interleaved scatter plot summarizing the alteration of MMPs shown in D. Data are represented as mean ± SEM and are representative of 3 independent experiments. Statistical significance was assessed by 1-way ANOVA. (F) Western blot of indicated proteins was performed in THP1 cells transduced with lentivirus expressing either shCtrl, shCD84 (3′-UTR), or shCD84 (3′-UTR) plus CD84-WT. Data are representative of at least 2 independent biological replicates.

Fig 5: CD84 knockdown deactivated energy metabolism and induced mitochondrial stress in AML.(A and B) Scattergrams of CD84-related gene sets based on enrichment analyses of DEGs in HEL cells (shCD84 vs shCtrl) (A) and THP1 cells (shCD84 vs shCtrl) (B). The color indicates the false discovery rate q values; NES, normalized enrichment score. (C) Venn diagram showing the overlapped DEGs between HEL (shCD84 versus shCtrl) and THP1 (shCD84 versus shCtrl) groups. (D) Heatmap showing gene expression of the overlapped differential genes between THP1 cells and HEL cells expressing shCD84 or shCtrl, based on a fold change >2 or <0.5 and P < 0.05. (E) Bar chart showing GO enrichment analysis of common DEGs (n = 188) in 2 AML cell lines. (F) Connecting lines showing the effects of CD84 deletion on levels of OCR and ECAR in THP1 cells. Cells were transfected with lentivirus expressing shCD84 or shCtrl, and puromycin selected for 2 days. The cells were harvested to measure levels of OCR and ECAR using the Seahorse XF Cell Energy Phenotype Test Kit. Data are represented as mean ± SEM and are representative of 3 biological replicates. Statistical significance was assessed with 2-way ANOVA (mixed model). (G) Box chart showing the effects of CD84 knockdown on FAO levels in THP1 cells. The cells were harvested as described in F, and FAO assay results are presented as fold change, compared with control. Data are represented as mean ± SEM and are representative of 3 independent experiments. Statistical significance was assessed by 2-tailed unpaired t test. (H) Connecting lines showing the effects of CD84 deletion on levels of OCR and ECAR in primary AML cells obtained from n = 3 different donors. Cells were transduced with lentivirus expressing shCD84 or shCtrl for 48 hours. The cells were harvested to measure levels of OCR and ECAR using the Seahorse XF Cell Energy Phenotype Test Kit. Data are represented as mean ± SEM and are representative of 3 independent experiments. Statistical significance was assessed with 2-way ANOVA (mixed model).