Vitamin D Receptor Expression in Plasmablastic Lymphoma and Myeloma Cells Confers Susceptibility to Vitamin D

Abstract

Plasmablastic B-cell malignancies include plasmablastic lymphoma and subsets of multiple myeloma and diffuse large B-cell lymphomaDLBCL. These diseases can be difficult to diagnose and treat, and they lack well-characterized cell line models. Here, immunophenotyping and FOXP1 expression profiling identified plasmablastic characteristics in DLBCL cell lines HLY-1 and SU-DHL-9, associated with CTNNAL1, HPGD, RORA, IGF1, and/or vitamin D receptor (VDR) transcription. We demonstrated VDR protein expression in primary plasmablastic tumor cells and confirmed in cell lines expression of both VDR and the metabolic enzyme CYP27B1, which catalyzes active vitamin D3 production. Although Vdr and Cyp27b1 transcription in normal B cells were activated by interleukin 4 (IL-4) and CD40 signaling, respectively, unstimulated malignant plasmablastic cells lacking IL-4 expressed both VDR and CYP27B1. Positive autoregulation evidenced intact VDR function in all plasmablastic lines, and inhibition of growth by active vitamin D3 was both dependent on MYC protein inhibition and could be enhanced by cotreatment with a synthetic ROR ligand SR-1078. Furthermore, a VDR polymorphism, FOK1, was associated with greater vitamin D3-dependent growth inhibition. In summary, HLY-1 provides an important model of strongly plasmablastic lymphoma, and disruption of VDR pathway activity may be of therapeutic benefit in both plasmablastic lymphoma and myeloma.

Figure 1.

Reduced FOXP1 expression and immunophenotyping identify plasmablastic lymphoma/myeloma cell lines. (a) Surface flow cytometry of DLBCL and myeloma cell lines, representative of n = 3. (b) Real-time polymerase chain reaction of B-cell (PAX5) and plasma cell (XBP1 and PRDM1) markers, relative to highest expressing line (100%), n = 3 ± SD. (c, left panels) Immunohistochemistry of FOXP1, CD138, CD20, PAX5, and IRF4, representative of two experiments. (c, right panels) Morphology of DLBCL and myeloma cell lines subjected to hematoxylin and eosin stain. SD, standard deviation.

Figure 2.

Expression of normal plasmablastic markers and VDR by DLBCL and myeloma cell lines. (a) Real-time polymerase chain reaction (PCR) of plasmablastic markers, relative to highest expressing line (100%), n = 3 ± SD. (b) Heat map showing Affymetrix probe set signals from DLBCL cell line gene expression profiling (32) identifying genes coding for cell surface proteins or with “receptor” annotation, having abundant expression in HLY-1 compared with OCI-Ly3 and OCI-Ly10, ranked by strength of expression in HLY-1. (c, upper panel) Real-time PCR of VDR transcript expression in lymphoma and myeloma lines relative to highest expressing line (100%), n = 3 ± SD, usingtwo independent probes. (c, lower panels) Immunoblotting of FOXP1, PAX5, and VDR in the same cell lines. (d) Immunoblotting of VDR in cells exponentially growing or arrested by either saturating culture (confluent) or treatment of 48 hours with 1 µM CDK4/6 inhibitor palbociclib; semiquantitative analysis of immunoblotting shown below as ratio of VDR to β-actin signal, mean ± SE of duplicate experiments. SD, standard deviation; SE, standard error.

Figure 3.

Primary plasmablastic lymphoma/myeloma tumor cells express nuclear VDR protein. Immunohistochemistry to detect VDR protein expression [antibody D-6 (Ab1) using a brown substrate, blue hematoxylin counterstain] in (a) cell lines, HBL-1 (negative, ABC-DLBCL), SU-DHL-9 (weakly plasmablastic DLBCL), HLY-1 (strongly plasmablastic ABC-DLBCL), and Thiel (plasmablastic myeloma), and in (b) primary plasmablastic tumors. Higher magnification in (A) right-hand panel highlights mitotic VDR expression.

Figure 4.

Primary plasmablastic lymphomas/myelomas express nuclear VDR protein associated with proliferative index. (a) Summary of VDR (antibody D-6) staining of primary lymphoma/myeloma cases. In some cases, sequential biopsy specimens from multiple presentations demonstrated maintained nuclear VDR expression throughout the disease course. Percentages indicate approximate proportion of tumor cells positively stained. (b) Nuclear VDR positivity was quantified approximately by multiplying stain intensity (–/±/+/++ as 0/1/2/3 respectively) by percentage of tumor cells stained and compared with percentage of Ki-67 positivity in tumor cells determined by clone MM1 immunohistochemistry.

Figure 5.

Vitamin D levels determine VDR expression in plasmablastic tumor cells. (a, left panels) Real-time polymerase chain reaction (PCR) of Vdr and Cyp27b1 transcripts in 24-hour stimulated murine naive B-cell cultures relative to T = 0 untreated sample, n = 2 ± SE. (a, center panel) Immunoblotting of Vdr and Cyp27b1 in parallel T = 0 untreated or 48-hour stimulated cultures. (a, right panel) Semiquantitative analysis of immunoblots, mean ± SE. (b) Real-time PCR of CYP27B1 and CYP27B1 immunoblotting in lymphoma and myeloma cell lines; real-time transcript expression relative to highest expressor. (c, left panel) VDR immunoblotting of cell lines treated 24 hours with 10⁻⁷ M VitD3 (+) or ethanol vehicle (–). (c, right panels) Schematic of VDR regulation in normal and plasmablastic cells. SE, standard error.

Figure 6.

VDR expression promotes viability and susceptibility to VitD3-induced cell cycle inhibition. (a, left panels) Number of trypan blue–positive (Dead) and –negative (Viable) HLY-1 and SU-DHL-9 cells after treatment with ethanol vehicle (–) or VitD3 (+, single 10⁻⁷ M dose at T = 0), n = 3 ± SD, and representative flow cytometry of BrdU incorporation/7-aminoactinomycin D (7-AAD) DNA content of cells cultured similarly for 48 hours. Numbers indicate mean percentage BrdU⁺ from n = 3 ± SD. (a, right panels) Immunoblotting of similarly treated HLY-1, representative of 2 experiments. (b) Analyses as performed in (a) on HLY-1 treated for 48 hours with 50 µM c-Myc inhibitor 10058-F4 or DMSO vehicle. (c) Real-time polymerase chain reaction of MYC and IL6 in HLY-1 treated for 24 hours with ethanol vehicle or indicated dose of VitD3, expressed as fold change relative to vehicle, n = 4 ± SD. (d) Representative immunoblotting and relative viable cell number determined by MTS assay 48 hours after transfection with control or one of two independent small interfering RNAs targeting VDR, n = 3 ± SD. SD, standard deviation. *P < 0.05, comparisons to same time-point vehicle or control siRNA sample.

Figure 7.

VitD3 effects on plasmablastic lymphoma cells can be enhanced by the synthetic ROR ligand SR-1078. (a, left panel) Relative viable cell number determined by MTS assay after 72-hour liquid culture with treatment indicated on right, n = 4 ± SD. (a, right panel) Colony number after 7 days of semisolid methylcellulose culture containing treatments indicated, initiated after 24-hour liquid culture pretreatment, n = 3 ± SE. (b) Flow cytometry of BrdU incorporation into, and 7-aminoactinomycin D (7-AAD) DNA content of, cells cultured 48 hours with treatments as indicated; numbers indicate mean percentage BrdU⁺, n = 3 ± SD. (c, left panels) Morphology of JJN-3 treated with vehicle or combined VitD3 plus SR-1078 (VitD3 + SR), after cytospin and hematoxylin and eosin stain. (c, upper right panels) Annexin V/propidium iodide staining of unfixed JJN-3 72 hours after vehicle or VitD3 + SR-1078 treatment; numbers represent mean percentage of cells within right top (late apoptotic) and right bottom (early apoptotic) quadrants, n = 3 ± SD. (c, lower right panels) Immunoblotting of cleaved poly ADP-ribose polymerase PARP in JJN-3 72 hours after treatment, representative of two experiments. SD, standard deviation; SE, standard error. *P < 0.05, ^▵P < 0.01, comparisons to vehicle-treated samples.