Nickerson, Helen

Abstract Approximately 15% of multiple myeloma (MM) is characterized by a t(4;14) translocation that causes the simultaneous dysregulation of MMSET on der(4) and fibroblast growth factor receptor 3 gene (FGFR3) on der(14). We reported several lines of evidence indicating a role for FGFR3 in myeloma tumorigenesis. First, activated FGFR3 is an oncogene capable of transforming fibroblasts. Second, FGFR3 activating mutations are acquired by MM cells during tumor progression. Third, targeted inhibition of FGFR3 leads to terminal differentiation and apoptosis in two t(4;14) MM cell lines. However, expression of FGFR3, but never of MMSET, is lost in about 25% of t(4;14) MM. Therefore, the overexpression of MMSET in all MM tumors with a t(4;14) translocation, and its homology to MLL, the oncogene on 11q23 translocated in acute leukemia suggest a critical role for MMSET in MM. To determine whether MMSET is an oncogene in vivo, we have generated transgenic mice in which MMSET expression in driven in lymphocytes by the lck proximal promoter juxtaposed to the Emu enhancer. Using the same expression vector we and others have obtained specific, high levels of transgene expression in B and T cells from spleen, bone marrow and thymus. Four transgenic lines were generated and although we detected MMSET expression in T cells in each of them, unexpectedly no expression in B cells was seen. This is consistent with our inability to ectopically express MMSET in B cell lines. Nevertheless B lymphoid tumors expressing MMSET developed at 23 month of age in each line (18/51 mice). Only 1/19 wild type matching control mice developed a splenomegaly. By Southern blot, monoclonal rearrangements of IgH, IgL and TCR β were detected within the same tumor population. In conclusion, this is the first report that MMSET is an oncogene capable of transforming lymphoid cells in an animal model. We are currently crossing these mice with FGFR3 transgenic mice to assess cooperation between these two oncogenes in tumorigenesis. Obviously a more restricted expression of MMSET in germinal center cells is required to investigate the role of MMSET in MM. Therefore, as we have done for c-myc, we are generating new transgenic mice in which MMSET expression will be activated sporadically in germinal center B cells by somatic hypermutation.

Abstract Dysregulation of D type cyclins is an almost universal event in multiple myeloma. Overexpression of cyclin D1 may occur by chromosomal translocation of t(11:14)(q13:32), and also in association with hyperdiploidy. To investigate the role of cyclin D1 in the growth of human myeloma cell lines, we used both shRNA plasmid constructs, and an siRNA Smartpool against cyclin D1 and compared to control cells transfected with anti-GFP or GAPDH constructs. siRNA was electroporated into multiple myeloma cell lines overexpressing cyclin D1 (KMS12PE, U266, H929) which gave a 50–60% transfection efficiency, assessed using a GFP plasmid. Successful reduction in levels of cyclin D1 was confirmed by western blotting and normalization to a beta actin standard. Cyclin D1 protein in sh- or siRNA transfected cells was reduced on average to 32% of that in cells transfected with an anti-GAPDH or anti-GFP siRNA. In individual experiments reduction of cyclin D1 protein to as little as 10% of control was observed. However, we found no sign of increased apoptosis by Annexin V/Propidium iodide staining. Furthermore, cell cycle analysis by ethidium bromide staining and flow cytometry revealed no significant change in the cell cycle of sh- or siRNA transfected cells when compared to control cells. qPCR analysis revealed no acute compensatory increase in the RNA levels of other D-type cyclins (D2, D3) following reduction of cyclin D1 levels. These results may be explained by the residual presence of sufficient cyclin D1 protein for cell cycle progression. An alternative explanation is that changes that frequently occur in myeloma to other cell cycle regulators, for example p16(INK4a) and Rb are able to circumvent cell cycle effects of reducing cyclin D1protein. These results suggest that the human myeloma cell lines tested are not acutely sensitive to cyclin D1 level.

Abstract The time in cellular differentiation at which the expression of an oncogene is dysregulated is thought to determine the phenotype of the resulting neoplasia. We tested this assumption in C57BL6/J transgenic mice expressing an HA-tagged c-myc oncogene in B cells under the control of kappa light chain regulatory elements. Two transgenes were engineered that differed by only one nucleotide. The first set of mice carries a wild type HA-MYC transgene, develops pro-B cell tumors, and succumbs rapidly to pronounced lymphosplenomegaly. In the second set of mice, a point mutation in the HA-tag portion of the transgene creates a stop codon that abrogates HA-MYC translation. This stop codon was engineered to be a hotspot for somatic hypermutation. Thus sporadically, in a germinal center B cell, somatic hypermutation may revert the stop codon, allowing translation of HA-MYC. In two independently derived lines, mice spontaneously develop monoclonal gammopathies (50% incidence at 30 weeks (n=35), 80% at 40 weeks (n=35)). Serum protein electrophoresis detects M-spikes that increase in intensity over time, and 6 out of 7 cases tested so far were of IgG1 isotype. Unlike other mouse models of plasma cell neoplasia, no lymphosplenomegaly nor ascites were detected. In analogy to human multiple myeloma, that is a disease of isotype-switched and hypermutated cells homing to the bone, large populations of plasma cells were found in the bone marrow. Bone marrow (but not spleen) lysates reacted for HA and human MYC proteins by western blot and immunohistochemistry, thus indicating that reversion of the stop codon occurred. Remarkably, in two young mice, monoclonal spikes appeared 2 weeks after vaccination with NP-CGG. These spikes were sustained and the monoclonal protein was reactive to the NP antigen. Together, comparison of the two sets of mice demonstrates that activation of the same transgene at distinct B cell developmental stages results in dramatically different tumor phenotypes. In addition, and opposite to most engineered mice, we have created a system where the oncogene is turned on sporadically, as occurs in the human disease. Furthermore, we have developed a model that faithfully reproduces important clinical aspects of monoclonal gammopathy of undetermined significance (MGUS) and its progression to multiple myeloma. This model will be useful to develop immunologic and chemoterapeutic approaches.

Abstract The B-Cell Lymphoma 6 (BCL6) gene encodes for a zinc finger motifs containing transcriptional repressor that is frequently dysregulated by chromosomal translocations in germinal center lymphomas. A putative protooncogene, its transforming ability in vivo was reported in I-mu-HA-BCL6 knock-in mice by Cattoretti et al last year. We also tested this assumption in transgenic mice expressing BCL6 in B cells under the control of kappa light chain regulatory elements. We replaced the murine C-kappa locus with the 16kb human BCL6 genomic locus in a construct containing the murine kappa light chain regulatory elements (Vk, EiK, 3′RR). While control transgenics were readily obtained (5/32 founders), only 3/68 founders were positive for the BCL6 transgene, of which only one (bearing a single copy of the transgene) was able to transmit the transgene to its progeny, thus suggesting embryonal toxicity of exogenous BCL6. In the bone marrow, flow cytometry revealed a nearly complete block of B cell development at the pro-B to pre-B transition. This was also the stage at which we first detected expression of EGFP in control reporter mice that were generated in parallel. Spleens of transgenic mice weighed about 50% of control spleens and less than 5% of splenocytes were CD19+ B cells. These were IgM high, IgD intermediate, corresponding to an immature B cell phenotype. Lymph nodes were smaller and B cells barely detected. Peyers’ patches were not visible. Combined, our analysis of 6–8 weeks old VkHABCL6 transgenic mice reveals that enforced expression of BCL6 early in development results in a profound block of B lymphocyte differentiation. How transgenic BCL6 modulates this effect at the transcriptional level remains to be investigated. To test the oncogenic potential of BCL6 in B cells, it will be interesting to precisely turn on this gene in the germinal center.