HTLV-1 viral oncogene HBZ induces osteolytic bone disease in transgenic mice

Abstract

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell malignancy that occurs in HTLV-1 infected patients. Most ATL patients develop osteolytic lesions and hypercalcemia of malignancy, causing severe skeletal related complications and reduced overall survival. The HTLV-1 virus encodes 2 viral oncogenes, Tax and HBZ. Tax, a transcriptional activator, is critical to ATL development, and has been implicated in pathologic osteolysis. HBZ, HTLV-1 basic leucine zipper transcription factor, promotes tumor cell proliferation and disrupts Wnt pathway modulators; however, its role in ATL induced osteolytic bone loss is unknown. To determine if HBZ is sufficient for the development of bone loss, we established a transgenic Granzyme B HBZ (Gzmb-HBZ) mouse model. Lymphoproliferative disease including tumors, enlarged spleens and/or abnormal white cell counts developed in two-thirds of Gzmb-HBZ mice at 18 months. HBZ positive cells were detected in tumors, spleen and bone marrow. Importantly, pathologic bone loss and hypercalcemia were present at 18 months. Bone-acting factors were present in serum and RANKL, PTHrP and DKK1, key mediators of hypercalcemia and bone loss, were upregulated in Gzmb-HBZ T cells. These data demonstrate that Gzmb-HBZ mice model ATL bone disease and express factors that are current therapeutic targets for metastatic and bone resident tumors.

Keywords: ATL; HBZ; HTLV-1; bone; leukemia.

Figure 1. Granzyme B HBZ mice develop lymphoproliferative disease

A. Granzyme B HBZ plasmid schematic and Gzmb-HBZ mouse. Arrow denotes a mesenchymal lymph node tumor in a Gzmb-HBZ mouse. B. HBZ mRNA expression in 4 month old WT and Gzmb-HBZ mice by Real-Time RT-PCR. C. Tumor incidence in 18-month WT and Gzmb-HBZ mice. D. Spleen weight in Gzmb-HBZ tumor free (n = 8) and tumor-bearing mice (n = 7). E. Percent of WT (n = 7) and Gzmb-HBZ (n = 15) mice with normal white blood cell counts (WBC). Normal WBC range was determined as the WT median +/- 2 standard deviations. B.-D. Statistical analysis represents mean +/- SEM. Statistical significance determined by non-parametric student’s t-test B., D. or Fisher’s exact test C., E. as appropriate. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 2. Lymphoid derived Gzmb-HBZ tumors express HBZ

A. Gzmb-HBZ tumor immunohistochemistry (IHC) for hematopoietic (CD45), T cells (CD3e) and B/activated T cells (B220) in a thoracic lymph node-derived tumor. Scale bar = 50 µm. B. Flow cytometry analysis of T cell populations in a Gzmb-HBZ derived mesenteric lymph node tumor. C. Flag-HBZ expression in tumor, spleen and tibia of Gzmb-HBZ mice by IHC. Scale bar = 20 µm. D. HBZ RNA in Gzmb-HBZ tumor cells by in situ hybridization, representative images and quantification. Scale bar = 50 µm.

Figure 3. T cells from Gzmb-HBZ mice are transplantable

A. Spleen weights from NSG mice implanted intravenously with PBS or mesenteric lymph node tumor cells (n = 5/group). B. Splenic CD4 and C. CD8 T cells from sham or tumor implanted mice. D. Image of a Gzmb-HBZ mouse 2-months post intraperitoneal tumor cell implantation with corresponding spleen weight E. and thymus weight F. *Red asterisk denotes a tumor. Percent of splenic CD3e T cells G. or CD4 and CD8 H. T cells from NSG mice serially implanted with tumor cells (D) or WT splenocytes (n = 5/group). All data reported as mean +/- SEM with statistical significance determined by Mann-Whitney U-test A. or unpaired t-test B. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 4. Gzmb-HBZ mice develop bone loss and hypercalcemia

A. Representative X-ray images of WT and Gzmb-HBZ tibiae. MicroCT analysis of bone volume/tissue volume (BV/TV) and trabecular thickness (Tb.Th) at 4 months of age (n = 5/group) B. and 18 months of age C. in WT (n = 7) and Gzmb-HBZ (n = 13) mice. D. MicroCT representative images. Scale bar = 300 µm. TRAP+ cells (arrowheads) E. and quantification of osteoclast surface per bone surface F. in WT (n = 5) and Gzmb-HBZ (n = 3) tibiae. Scale bar = 50 µm. G. Enzyme-linked immunosorbent assay (ELISA) for osteoblast activity marker Procollagen Type I Intact N-terminal Propeptide (P1NP), measured in serum from Gzmb-HBZ (n = 4) and WT (n = 7) mice. H. Serum calcium levels in WT (n = 5) and Gzmb-HBZ (n = 4) mice at 18 months. All data reported as mean +/- SEM. Statistical significance was determined by unpaired t-test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 5. Gzmb-HBZ mice have systemic inflammation and increased T-cell derived bone-acting factors

A. ELISA quantification of serum cytokines in 18-month WT (n = 3) and Gzmb-HBZ (n = 3) mice. Flow cytometry analysis of ex vivo expanded T cell populations B. and IFNγ expression C. from WT and Gzmb-HBZ splenocytes. Granzyme B D., HBZ E., PTHrP F., RANKL G. and DKK1 H. mRNA expression in WT and Gzmb-HBZ ex vivo cultured, activated T cells. Data is representative of 2-3 biological replicates. All data reported as mean +/- SEM. Statistical significance determined by unpaired t-test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.