The IMiDs targets IKZF-1/3 and IRF4 as novel negative regulators of NK cell-activating ligands expression in multiple myeloma

Abstract

Immunomodulatory drugs (IMiDs) have potent anti-tumor activities in multiple myeloma (MM) and are able to enhance the cytotoxic function of natural killer (NK) cells, important effectors of the immune response against MM. Here, we show that these drugs can enhance the expression of the NKG2D and DNAM-1 activating receptor ligands MICA and PVR/CD155 in human MM cell lines and primary malignant plasma cells. Depletion of cereblon (CRBN) by shRNA interference strongly impaired upregulation of these ligands and, more interestingly, IMiDs/CRBN-mediated downregulation of the transcription factors Ikaros (IKZF1), Aiolos (IKZF3) and IRF4 was critical for these regulatory mechanisms. Indeed, shRNA knockdown of IKZF1 or IKZF3 expression was both necessary and sufficient for the upregulation of MICA and PVR/CD155 expression, suggesting that these transcription factors can repress these genes; accordingly, the direct interaction and the negative role of IKZF1 and IKZF3 proteins on MICA and PVR/CD155 promoters were demonstrated. Finally, MICA expression was enhanced in IRF4-silenced cells, indicating a specific suppressive role of this transcription factor on MICA gene expression in MM cells.Taken together, these findings describe novel molecular pathways involved in the regulation of MICA and PVR/CD155 gene expression and identify the transcription factors IKZF-1/IKZF-3 and IRF4 as repressors of these genes in MM cells.

Keywords: DNAM-1Ls; IMiDs; NKG2DLs; multiple myeloma; natural killer.

Figure 1. IMiDs upregulate MICA and PVR/CD155 expression on human Multiple myeloma cells and enhance their recognition by NK cells

A. MICA, MICB and PVR/CD155 surface expression were analyzed by flow cytometry on SKO-007(J3) cells treated with lenalidomide (Lena) (10 μM) for 72h. The grey colored histograms represent basal expression of the indicated ligand, while thick black histograms represent the expression after treatment with the drug. Data are representative of one out of four independent experiments. B. The MFI of MICA, MICB and PVR/CD155 were calculated based on at least four independent experiments and evaluated by paired Student t test (*P < 0.05). Histograms represent the MFI of specific mAb - MFI of isotype control. C. NK cells, prepared from PBMCs of healthy donors, were incubated with SKO-007(J3) cells, untreated or treated with lenalidomide (Lena) for 72h, and used as target cells in a degranulation assay. The assay was performed at the effector:target (E:T) ratio of 2.5:1. After 3 hours at 37°C, cells were stained with anti-CD56, anti-CD3 and anti-CD107a mAbs. Cell surface expression of CD107a was analyzed on FSC/SSC-gated and CD56⁺CD3⁻ cells. In order to evaluate the role of NKG2D and DNAM-1, the assay was performed in parallel treating NK cells with blocking anti-DNAM-1 or anti-NKG2D antibodies. The MFI of CD107a were calculated based on at least three independent experiments and evaluated by paired Student t test (*P < 0.05). For each treatment, histograms represent the MFI of specific mAb - MFI of isotype control. D. CD138⁻ bone marrow cells, cultured for 2 days in complete medium supplemented with IL-2 (200 U/mL), were incubated with purified autologous myeloma cells, untreated or treated with lenalidomide (Lena) for 48h, and used as target cells in a degranulation assay. The assay was performed at the effector:target (E:T) ratio of 2.5:1. After 3 hours at 37°C, cells were stained with anti-CD56, anti-CD3 and anti-CD107a mAbs. Cell surface expression of CD107a was analyzed on CD56⁺CD3⁻ cells. Results obtained from two patients (P11 and P12) are represented.

Figure 2. Lenalidomide increases MICA and PVR/CD155 mRNA expression and promoter activation in SKO-007(J3) cells

A. Real Time PCR analysis of total mRNA obtained from SKO-007(J3) cells, untreated or treated with lenalidomide (Lena) as described above for 24h and 48h. Data, expressed as fold change units, were normalized with GAPDH and referred to the untreated cells, considered as calibrator and represent the mean of 3 experiments (*P < 0.05). B. Real Time PCR analysis of total mRNA obtained from purified CD138⁺cells untreated or treated with lenalidomide (Lena) for 24h in complete medium supplemented with 20 ng/ml IL-3 and 2 ng/ml IL-6. Data, expressed as fold change units, were normalized with GAPDH and referred to the untreated cells considered as calibrator. C. SKO-007(J3) cells were infected with lentivirus encoding LV control, LV-MICA or LV-PVR/CD155 promoter, obtained as described in Materials and Methods. After 48h treatment with lenalidomide (Lena), cellular medium were harvested and analyzed for the Gaussia Luciferase and SEAP assays. Results are expressed as relative luciferase activity normalized to SEAP activity and represent the mean value from four independent experiments (*P < 0.05).

Figure 3. MICA and PVR/CD155 upregulation by IMiDs is CRBN-dependent and involves IKZF1/3 transcription factors

A. Total mRNA was obtained from SKO-007(J3) cells transiently infected with lentivirus pLKO-shRNA-CRBN or pLKO non-targeting shRNA (control) (72h) and analyzed for CRBN mRNA expression by Real-Time qRT-PCR. Data were normalized with GAPDH and referred to the cells infected with non-target shRNA, considered as calibrator. B. MICA, MICB and PVR/CD155 surface expression were analyzed by flow cytometry on SKO-007(J3) pLKO non-target shRNA or pLKO-shRNA-CRBN cells, treated with lenalidomide (Lena) as described above. The grey colored histograms represent basal expression of the indicated ligand, while thick black histograms represent the expression after treatment with the drug. Data are representative of one out of five independent experiments. C. The MFI of MICA, MICB and PVR/CD155 were calculated based on at least five independent experiments and evaluated by paired Student t test (*P < 0.05). For each treatment, histograms represent the MFI of specific mAb - MFI of isotype control. D., F. MICA, MICB and PVR/CD155 surface expression were analyzed by flow cytometry on pLKO-IKZF1/GFP or F., G.) pLKO-IKZF3/GFP -lentivirus transiently infected SKO-007(J3) cells (72h), by gating on GFP⁺ and GFP⁻ cells as indicated in Suppl. Fig. 7C. Data are representative of one out of four independent experiments. The grey colored histograms represent the expression of the indicated ligand in GFP⁻ cells while thick black histograms represent the expression of the indicated ligand in GFP⁺ infected cells. E., G. The MFI of MICA, MICB and PVR/CD155 were calculated based on at least four independent experiments and evaluated by paired Student t test (*P < 0.05). For each treatment, histograms represent the MFI of specific mAb - MFI of isotype control.

Figure 4. IKZF1/3 transcription factors repress mica and pvr promoter activity

A., C. 293T cells were transiently co-transfected with the indicated −270bp-mica reporter vectors or different pvr promoter deletions and an IKZF1 expression vector as described in Materials and Methods. After 48h, cells were harvested and protein extracts were prepared for the luciferase assay. Data were normalized to protein concentration and β-galactosidase activity and represent the mean of three independent experiments (*P < 0.05). B., D. In vivo binding of IKZF1 and IKZF3 to the MICA and PVR promoters was examined in ChIP analysis. Samples were immunoprecipitated from sonicated lysates of SKO-007(J3) cells using an antibody against human IKZF1 or IKZF3 proteins or isotype control. After reversing the cross-linking, DNA was precipitated and Real-Time PCR was performed using primers to amplify the mica or pvr promoter region encompassing the Ikaros responsive element (IK-RE) (indicated in the figures). Results are expressed as Relative Enrichment as compared to the input. Data represent the mean of n=3 experiments (*P < 0.05). IK-RE: Ikaros Responsive Element.

Figure 5. A mutant form of Ikaros incapable of binding CRBN abrogates MICA and PVR/CD155 upregulation by IMiDs

A. Western Blot analysis of total cellular proteins from SKO-007(J3) cells infected with a control empty Lenti-Ires-GFP vector or Lenti-Ires-GFP-IKZF1-V1-Q146H, left unstimulated or stimulated with lenalidomide (Lena) for 72h. The proteins transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of proteins had been loaded in each lane and immunoblotted for IKZF1 and Actin. B. MICA, MICB and PVR/CD155 surface expression were analyzed by flow cytometry on SKO-007(J3) cells transiently infected with lentiviral vectors encoding GFP or GFP and IKZF1-V1-Q146H, untreated or treated with lenalidomide (Lena) as described above. Data are representative of one out of five independent experiments. The grey colored histograms represent basal expression of the indicated ligand, while thick black histograms represent the expression after treatment with lenalidomide (Lena). C. The MFI of MICA, MICB and PVR/CD155 were calculated based on at least five independent experiments and evaluated by paired Student t test (*P < 0.05). For each treatment, histograms represent the MFI of specific mAb - MFI of isotype control.

Figure 6. IRF4 represses MICA expression

A. Lysates of SKO-007(J3) cells untreated or treated with lenalidomide (Lena) for 24h, 48h and 72h were subjected to Western Blotting using anti-IRF4 and Actin antibodies. The proteins transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of proteins had been loaded in each lane. Data are representative of one out of three independent experiments. B. Immunoblotting analysis for IRF4 and Actin of total cellular proteins from SKO-007(J3) cells infected with two different lentiviruses, pLKO expressing IRF4-shRNA or pLKO non-targeting shRNA (control) for 72h. The proteins transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of proteins had been loaded in each lane. Data are representative of one out of three independent experiments. C. MICA, MICB and PVR/CD155 surface expression were analyzed by flow cytometry on pLKO-control (non-targeting) or pLKO-IRF4-lentivirus infected SKO-007(J3) cells (72h). Data are representative of one out of four independent experiments. The grey colored histograms represent the expression of the indicated ligand in cells transduced with the pLKO-control, while thick black histograms represent the expression in cells infected with pLKO-IRF4. D. Total RNA were also isolated from infected cells for Real-Time qRT-PCR analysis. Data, expressed as fold change units, were normalized with GAPDH and referred to the cells infected with non-target shRNA, considered as calibrator and represent the mean of 3 experiments (*P < 0.05). E. NK cells prepared from PBMCs of healthy donors were incubated with SKO-007(J3) cells after 72h-infection with pLKO-control (non-targeting) or pLKO-IRF4 shRNA and used as target cells in a degranulation assay. The assay was performed as described above. Results are representative of one out of three independent experiments.

Figure 7. MICA inhibition by IRF4 depends on its DNA binding activity

A. MICA and PVR/CD155 surface expression were analyzed by flow cytometry on SKO-007(J3) cells transduced with a retrovirus expressing IRF4 (1-405) or IRF4 (403-1356) and GFP, after 72h treatment with lenalidomide (Lena). Data are representative of one out of three independent experiments. The grey colored histograms represent basal expression of the indicated ligand, while thick black histograms represent the expression after treatment with the drug in GFP positive cells. B. The MFI of MICA, MICB and PVR/CD155 were calculated based on at least four independent experiments and evaluated by paired Student t test (*P < 0.05). For each treatment, histograms represent the MFI of specific mAb - MFI of isotype control.

Figure 8. Loss of the transcription factors IKZF1/3 and IRF4 contributes to IMiDs-mediated MICA upregulation with different kinetics

A., B. Real Time PCR analysis of total mRNA obtained from SKO-007(J3) cells, unstimulated or treated with lenalidomide (Lena) for the indicated times. Data, expressed as fold change units, were normalized with GAPDH and referred to the untreated cells considered as calibrator and represent the mean of 3 experiments (*P < 0.05). C. SKO-007(J3) cells were treated with lenalidomide (Lena) for the indicated times and cell lysates were immunoblotted for IRF4, IKZF1, IKZF3 and Actin. The proteins transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of proteins had been loaded in each lane. Data are representative of one out of three independent experiments. D. Immunoblotting analysis for IRF4, Blimp-1, IKZF1, IKZF3 and Actin of total cellular proteins from SKO-007(J3) cells infected with a lentivirus expressing IRF4 shRNA or pLKO-control (non-targeting) shRNA. The proteins transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of proteins had been loaded in each lane. Data are representative of one out of three independent experiments.