A novel rapid-onset high-penetrance plasmacytoma mouse model driven by deregulation of cMYC cooperating with KRAS12V in BALB/c mice

Abstract

Our goal is to develop a rapid and scalable system for functionally evaluating deregulated genes in multiple myeloma (MM). Here, we forcibly expressed human cMYC and KRAS12V in mouse T2 B cells (IgM(+)B220(+)CD38(+)IgD(+)) using retroviral transduction and transplanted these cells into lethally irradiated recipient mice. Recipients developed plasmacytomas with short onset (70 days) and high penetrance, whereas neither cMYC nor KRAS12V alone induced disease in recipient mice. Tumor cell morphology and cell surface biomarkers (CD138(+)B220(-)IgM(-)GFP(+)) indicate a plasma cell neoplasm. Gene set enrichment analysis further confirms that the tumor cells have a plasma cell gene expression signature. Plasmacytoma cells infiltrated multiple loci in the bone marrow, spleen and liver; secreted immunoglobulins; and caused glomerular damage. Our findings therefore demonstrate that deregulated expression of cMYC with KRAS12V in T2 B cells rapidly generates a plasma cell disease in mice, suggesting utility of this model both to elucidate molecular pathogenesis and to validate novel targeted therapies.

Figure 1

cMYC/KRAS12V can transform BaF3 cells independent of IL3 and transduce T2 B cells in vitro. (a) Workflow for generation of the adoptive plasmacytoma mouse model. (b) Schematic diagram of MSCV-based retroviral vectors: MIG, cMYC, KRAS12V and cMYC/KRAS12V. (c) Western blot analysis showed expression of MIG, cMYC, KRAS12V and cMYC/KRAS12V in transfected 293T cells. Total protein was analyzed against human MYC (upper) and RAS (middle); GAPDH (lower) served as a loading control. (d) KRAS12V and cMYC/KRAS12V drove BaF3 cells to grow independent of IL3 in vitro. Representative cells from two independent experiments are shown. (e) The purification of mouse IgM⁺ spleen B cells after micro-bead isolation is shown (left panel). The cell population transited to T2 B-cell subset (IgM⁺B220⁺ CD38⁺IgD⁺) after stimulation with LPS and mIL4 for 48 h (right three panels). Assays were independently and repeatedly performed. (f) Colonies in methylcellulose assays were observed only in cells transduced by cMYC/KRAS12V but not other groups. Three independent experiments were performed.

Figure 2

cMYC/KRAS12V-induced plasmacytoma in BALB/c mice. (a) Recipients receiving MIG- or cMYC- or KRAS12V-transfected donor cells remained tumor free, whereas all recipients of cMYC/KRAS12V cells died with plasmacytomas. Secondary transplantation recipients of cMYC/KRAS12V-transduced tumor cells died with similar syndromes (2nd, secondary transplantation). Primary transplantation was repeated five times, and secondary transplantation was independently and repeatedly performed. Group sizes and survival times are indicated. (b) Tumor in peritoneal cavity and splenomegaly were observed in the cMYC/KRAS12V group animals (n=20, from five independent experiments). (c) H&E staining showing tumor cell morphology (n=6). (d) Tumor cells from peritoneal cavity (P.C.) and ascites (A.C.) (n=10) were characterized as CD138⁺B220⁻IgM⁻GFP⁺ by flow cytometry.

Figure 3

Plasmacytoma cells infiltrated multiple organs. (a) Flow cytometry analysis of GFP and CD138 to track tumor cells in peripheral blood (PB), femur and tibia BM and spleen (SPL). Numbers represent tumor cell percentage in respective gates. Samples (n=25) of individual mice from different experiments were analyzed. (b) BM and spleen (SPL) sections from mice receiving MIG-, cMYC-, KRAS12V- and cMYC/KRAS12V-transfected cells were stained with hematoxylin-eosin (H&E) and anti-CD138 antibody to identify plasmacytoma cells. (c) Liver (LV) sections from diseased mice were stained with H&E (left) and anti-CD138 antibody (right). (d) IHC analysis of BM sections showed expression of cMYC, KRAS and eGFP in cMYC/KRAS12V-induced plasmacytoma cells.

Figure 4

Evidence of clonality and hypermutation of expressed Ig genes in cMYC/KRAS12V-induced plasmacytoma mice. (a) Serum protein electrophoresis (SPEP) in serum and ascites from cMYC/KRAS12V-induced plasmacytoma mice. The bands are indicated with arrows. Samples were obtained from all the group animals as labeled (n=5 each group). (b) Ig isoform identification. Ascites were collected from plasmacytoma mice and analyzed with Thermo Scientific Pierce Rapid ELISA Mouse mAb Isotyping Kit. The Ig isoforms (n=10) are represented by color labeled bands as indicated. (c) Southern blot analysis for clonotypic IgH -chain rearrangement in plasmacytoma cells. Genomic DNA was isolated from IgM⁺ B cells (sample 1) or cMYC/KRAS12V-induced plasmacytoma cells (samples 2–7), digested with EcoRI restriction enzyme and hybridized with murine JH4 probe. GL denotes germline band.

Figure 5

Renal and bone alterations in cMYC/KRAS12V-induced plasmacytoma mice. (a) Renal tissue from mice receiving MIG-, cMYC-, KRAS12V- and cMYC/KRAS12V-transfected cells (n=3 each group) were analyzed by light microscopy (hematoxylin-eosin staining) and IHC staining with antibodies against mouse Ig κ chains. (b) Histological sections of kidneys of mice receiving cMYC/KRAS12V-transduced plasmacytomas were analyzed by IHC staining with antibodies against mouse Ig κ and λ light chains, as well as IgG and IgM heavy chains. (c) Micro-CT analysis demonstrated no bone lesions in all the group animals. Representative graphs from three individual animals are shown.

Figure 6

Plasma cell transcription signatures in cMYC/KRAS12V-transduced plasmacytoma cells. (a) Expression of selected genes from microarray results were confirmed using quantitative real-time PCR. All data represent mean of triplicate experiments. (b) GSEA showed a plasma cell gene expression signature of plasmacytoma cells. (c) GSEA showed genes associated with B-cell receptor signaling to be significantly downregulated in plasmacytoma cells. (d) GSEA showed genes in IRF4 target gene set were upregulated in plasmacytoma cells. (e) Table of the gene sets related to plasma cell differentiation and survival.

Figure 7

Gene expression signatures of cMYC/KRAS12V-transduced plasmacytoma cells. (a) Using GSEA, four gene sets associated with MYC activation are presented in plasmacytoma cells compared with IgM⁺ B cells. (b) GSEA showed genes upregulated by mTOR in plasmacytoma cells. (c) GSEA showed that genes regulating cell cycle are significantly enriched in plasmacytoma cells. (d) Table of gene sets of MYC, mTOR, cell cycle and cancer-related pathways (from top to bottom) in cMYC/KRAS12V-transduced plasmacytoma cells.

Figure 8

Provirus integration sites. (a)The splinkerette-PCR results of provirus integration sites. The lanes of 3% agarose gel showed the splinkerette-PCR production for three mouse plasmacytoma samples with 100 bp marker. Each band represents a provirus integration site and a tumor cell subclone, except for the top band, which represents retroviral vector endogenous fragment (617 bp, not including the linker sequence). (b) Identification of MSCV integration sites in plasmacytoma cells.