FTY720 increases CD74 expression and sensitizes mantle cell lymphoma cells to milatuzumab-mediated cell death

Abstract

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a short median survival despite multimodal therapy. FTY720, an immunosuppressive drug approved for the treatment of multiple sclerosis, promotes MCL cell death concurrent with down-modulation of phospho-Akt and cyclin D1 and subsequent cell-cycle arrest. However, the mechanism of FTY720-mediated MCL cell death remains to be fully clarified. In the present study, we show features of autophagy blockage by FTY720 treatment, including accumulation of autolysosomes and increased LC3-II and p62 levels. We also show that FTY720-induced cell death is mediated by lysosomal membrane permeabilization with subsequent translocation of lysosomal hydrolases to the cytosol. FTY720-mediated disruption of the autophagic-lysosomal pathway led to increased levels of CD74, a potential therapeutic target in MCL that is degraded in the lysosomal compartment. This finding provided rationale for examining combination therapy with FTY720 and milatuzumab, an anti-CD74 mAb. Treatment of MCL cell lines and primary tumor cells with FTY720 and milatuzumab resulted in statistically significant enhanced cell death, which was synergistic in blastic variant MCL cell lines. Significant in vivo therapeutic activity of combination treatment was also demonstrated in a preclinical, in vivo model of MCL. These findings support clinical evaluation of this combination in patients with MCL.

Figures

Figure 1

FTY720 treatment blocks autophagy in MCL cells. (A) Four MCL cell lines were treated with FTY720 (Jeko-1, 12.5μM; Mino, 7.5μM; UPN-1, 12.5μM; and Z-138, 7.5μM), chloroquine (40μM), rapamycin (10μM), or combinations, harvested at 24 hours, and immunoblotted for the microtubule-associated protein light-chain 3 (LC3-I and LC3-II). Actin was used as a loading control. Representative histograms summarizing 3 independent experiments are also shown. Histograms were obtained using densitometry data for LC3-II levels in treated samples relative to untreated samples and normalized to the actin control. (B) The amount of total cellular LC3 was determined by confocal microscopy. Jeko-1, Mino, UPN-1, and Z-138 cells were treated with FTY at the indicated doses, chloroquine (40μM), rapamycin (10μM), or the combination of FTY720 and chloroquine for 4, 8, and 24 hours. LC3 fluorescence intensity was measured in 4 microscopic fields and integrated intensity was averaged relative to the number of cells per field (approximately 180-220 cells per condition). Representative histograms summarizing LC3 fluorescence intensity are shown. P values were calculated comparing FTY720, chloroquine, and rapamycin treatment with the untreated control. (C) Jeko-1, Mino, UPN-1, and Z-138 cells were treated with FTY720 at the doses indicated in panel A, chloroquine (40μM), rapamycin (10μM), or combinations, harvested at 24 hours, and immunoblotted for p62. Actin was used as loading control. Levels of p62 normalized with actin are also shown in panel C. Representative histograms summarizing 3 independent experiments are also shown. Histograms were obtained using densitometry data for p62 levels in treated samples relative to untreated samples and normalized to the actin control. (D) Representative TEM images showing ultrastructural changes observed with Jeko-1 and Mino cells treated with or without FTY720 at the indicated concentrations for 8 hours. The arrows indicate accumulation of autophagic vacuoles containing cytoplasmic material after exposure to FTY720 or chloroquine.

Figure 2

FTY720-mediated cell death is dependent on lysosomal membrane permeabilization and cathepsin activity. (A) To determine the relationship between lysosomal volume and cell death, Jeko-1 and Mino cells were treated with FTY720 or chloroquine at the indicated concentrations for 8 hours. Cells were then labeled with LysoTracker Green and costained with annexin V-PE. Changes in lysosomal volume (FL1) and cell death (FL2) were assessed by 2-channel flow cytometry. Representative histograms summarizing the percentage of LysoTracker+/annexin V+ cells are also shown. (B) Jeko-1 and Mino cells were treated with FTY720, chloroquine, rapamycin, or combinations at the indicated concentrations for 8 hours. Cells where then stained with AO (1 μg/mL) for 15 minutes. The relative changes in FL1 fluorescence were assessed by flow cytometry. Flow cytometric data from single experiments are shown in the left panels; representative histograms summarizing AO fluorescence intensity of MCL treated with FTY720, chloroquine, rapamycin, or combinations are shown in the right panels (MFI of treated cells is normalized to the untreated controls). (C) Jeko-1 and Mino cells were treated with FTY720 at the indicated concentration in the presence or absence of cathepsin inhibitor III (5 and 10μM). Cell death was determined by annexin V/propidium ioidie staining and flow cytometry at 8 hours. Data are shown as the percentage of annexin V−/propidium iodide− cells (live cells).

Figure 3

CD74 expression in FTY720-treated MCL cell lines. (A) Jeko-1 and Mino cell lines were treated with FTY720, chloroquine, rapamycin, or combinations at the indicated concentrations, harvested at 24 hours, and immunoblotted for CD74 (top panels). Actin was used as loading control. Representative histograms summarizing 3 independent experiments are also shown (middle panels). Histograms were obtained using densitometry data for CD74 levels in treated samples relative to untreated samples and normalized to the actin control. CD74 mRNA expression in MCL cell lines treated with FTY720 at the indicated concentrations for 8 or 24 hours was measured by real-time RT-PCR (bottom panels). The bar graph shows normalized fold expression of CD74 mRNA relative to untreated controls using GAPDH as an internal control. (B) CD74 MFI of MCL cells treated with FTY720, chloroquine, or rapamycin at the indicated concentrations after 8 and 24 hours. Cells were stained with an anti-CD74 Ab (FITC conjugated) and CD74 MFI was measured by flow cytometry. Flow cytometric data from single experiments are shown in the top panels; representative histograms summarizing the MFI of untreated and treated Jeko-1, Mino, UPN-1, and Z-138 cells are shown in the bottom panels. (C-D) Binding, internalization, and CD74 fluorescence intensity in Jeko-1 and Mino cells were examined by laser-scanning confocal microscopy. MCL cells were incubated with 5 μg/mL of rhodamine-conjugated milatuzumab (red) in the absence (C) or presence (D) of FTY720 at the indicated concentrations for 8 hours at 37°C. MCL cells were also stained with a primary Ab (anti-LC3) and a secondary Ab (Alexa Fluor 488–conjugated, green). DAPI was used for nuclear staining (blue). (E) The amount of total cellular CD74 was determined by confocal microscopy. Jeko-1, Mino, UPN-1, and Z-138 cells were treated with FTY720 at the indicated doses, chloroquine (40μM), rapamycin (10μM), or the combination of FTY720 and chloroquine for 4, 8, and 24 hours. CD74 fluorescence intensity was measured in 4 microscopic fields and integrated intensity was averaged relative to the number of cells per field (approximately 180-220 cells per condition). Representative histograms summarizing CD74 fluorescence intensity are shown. P values were calculated comparing FTY720, chloroquine, and rapamycin treatment with the untreated controls.

Figure 4

FTY720 sensitizes MCL cell lines and primary patient tumor cells to milatuzumab-mediated cytotoxicity. (A) Four MCL cell lines and primary cells from 6 patients were treated with FTY720 and/or milatuzumab plus cross-linking Abs at indicated the concentrations. (B) Patient characteristics. (C) Individual patient responses. (D) Representative histograms summarizing patient responses. Cell death was determined by annexin V/propidium iodide staining and flow cytometry at 24 hours. Data are shown as the percentage of annexin V−/propidium iodide− cells (live cells) and are normalized to untreated controls. Combination treatment resulted in a statistically significant enhanced induction of cell death compared with either agent alone in MCL cell lines and primary cells (P < .01). Combination treatment resulted in synergistic cell death in blastic variant–derived cell lines (Jeko-1, UPN-1, and Z-138), and was additive in a classic variant–derived cell line (Mino) and in primary MCL cells.

Figure 5

Evaluation of in vivo therapeutic activity of FTY720 and milatuzumab in the preclinical MCL model. SCID mice were injected intravenously with 40 × 106 JeKo cells and observed daily for signs of tumor burden. The median survival for FTY720- and milatuzumab-treated mice (n = 10) was 36 days (95% CI, 31-36) compared with 31 days for the FTY720-treated mice (95% CI, 28-32) and 33.5 days for the milatuzumab-treated mice (95% CI, 23-34).