Dasatinib therapy results in decreased B cell proliferation, splenomegaly, and tumor growth in a murine model of lymphoma expressing Myc and Epstein-Barr virus LMP2A.

Epstein-Barr virus (EBV) infection and latency has been associated with malignant diseases including nasopharyngeal carcinoma, Hodgkin lymphoma, Burkitt lymphoma, and immune deficiency associated lymphoproliferative diseases. EBV-encoded latent membrane protein 2A (LMP2A) recruits Lyn and Syk kinases via its SH2-domain binding motifs, and modifies their signaling pathways. LMP2A transgenic mice develop hyperproliferative bone marrow B cells and immature peripheral B cells through modulation of Lyn kinase signaling. LMP2A/λ-MYC double transgenic mice develop splenomegaly and cervical lymphomas starting at 8 weeks of age. We reasoned that targeting Lyn in LMP2A-expressing B cells with dasatinib would provide a therapeutic option for EBV-associated malignancies. Here, we show that dasatinib inhibits B cell colony formation by LMP2A transgenic bone marrow cells, and reverses splenomegaly and tumor growth in both a pre-tumor and a syngeneic tumor transfer model of EBV-associated Burkitt lymphoma. Our data support the idea that dasatinib may prove to be an effective therapeutic molecule for the treatment of EBV-associated malignancies.

A distinct expression of various gene subsets in CD34+ cells from patients with early and advanced myelodysplastic syndrome.

Gene expression profiles of CD34+ cells were compared between 51 MDS patients and 7 controls. The most up-regulated genes in patients included HBG2, HBG1, CYBRD1, HSPA1B, ANGPT, and MYC, while 13 genes related to B-lymphopoiesis showed down-regulation. We observed in advanced MDS patients decreased expression of genes involved in cell cycle control, DNA repair and increased expression of proto-oncogenes, angiogenic and anti-apoptic genes. The results suggest that increased cell proliferation and resistance to apoptosis together with a loss of cell cycle control, damaged DNA repair and altered immune response may play an important role in malignant clone expansion in MDS.

Novel pluripotential neural progenitor lines exhibiting rapid controlled differentiation to neurotransmitter receptor-expressing neurons and glia.

The immortalization of progenitor cells from embryonic murine hippocampus using oncogene-carrying retroviral vectors is described. Use of a vector encoding the oncogene v-myc results in lines of nestin-positive progenitor cells. Limited differentiation ensues if the cells are cultured in the presence of dibutyryl cyclic adenosine monophosphate. In contrast, use of a vector in which the extracellular portion of the epidermal growth factor (EGF) receptor is fused to the neu tyrosine kinase generates lines of pluripotential nestin-positive progenitor cells, which differentiate upon withdrawal of EGF into neurons and glia. Differentiated neurons expressing action potentials and neurotransmitter receptors make up a high proportion of the cells. These cell lines are useful tools to investigate the characteristics of differentiating neurons and glia, as well as to screen neuroactive drugs. This work has been reported in preliminary form as an abstract (1996 Society for Neuroscience Abstract, #606.20, p. 1537).

Bipotent progenitor cell lines from the human CNS.

Human central nervous system (CNS) cell lines would substantially facilitate drug discovery and basic research by providing a readily renewable source of human neurons. We isolated clonal human CNS cell lines that had been immortalized with a tetracycline (Tc)-responsive v-myc oncogene; addition of Tc to the growth medium suppressed the oncoprotein rapidly and virtually completely, allowing differentiation to proceed. Two classes of bipotent precursor cells were immortalized: the first class had a default differentiation pathway of neurons only, and the second class had a default differentiation pathway of neurons and astrocytes. We found that after exposure to different external signals in vitro, the environment is capable of redirecting the fate of a particular cell, even in the case of the bipotent precursor cell whose default differentiation pathway was neurons only. These data suggest that extrinsic cues can prevail over intrinsic determinants in directing cell fate in the human CNS.

Comparative analysis of the structure and function of the chicken c-myc and v-myc genes: v-myc is a more potent inducer of cell proliferation and apoptosis than c-myc.

To gain a more complete understanding of c-myc regulation in chickens, we have completed the structural characterization of the chicken c-myc gene and have begun to investigate c-myc transcription and protein expression. A comparison of c-myc structure and expression between mammals and birds presents an enigma: there are striking similarities in the pattern of gene expression in the absence of obvious sequence similarities in the controlling elements. We have begun to investigate c-myc and v-myc function using retroviral vectors that differ solely in the Myc proteins that they express. We show that while the overexpression of the smaller c-Myc protein is sufficient to induce morphological transformation in chicken embryo fibroblasts, overexpression of v-Myc provides a stronger signal for cells to enter the cell cycle and is a more potent inducer of apoptosis than c-Myc.

Differentiation of the immortalized adult neuronal progenitor cell line HC2S2 into neurons by regulatable suppression of the v-myc oncogene.

A regulatable retroviral vector in which the v-myc oncogene is driven by a tetracycline-controlled transactivator and a human cytomegalovirus minimal promoter fused to a tet operator sequence was used for conditional immortalization of adult rat neuronal progenitor cells. A single clone, HC2S2, was isolated and characterized. Two days after the addition of tetracycline, the HC2S2 cells stopped proliferating, began to extend neurites, and expressed the neuronal markers tau, NeuN, neurofilament 200 kDa, and glutamic acid decarboxylase in accordance with the reduced production of the v-myc oncoprotein. Differentiated HC2S2 cells expressed large sodium and calcium currents and could fire regenerative action potentials. These results suggest that the suppression of the v-myc oncogene may be sufficient to make proliferating cells exit from cell cycles and induce terminal differentiation. The HC2S2 cells will be valuable for studying the differentiation process of neurons.

The transcription activation domains of v-Myc and VP16 interact with common factors required for cellular transformation and proliferation.

The amino terminus of the avian myelocytomatosis virus MC29 v-Myc oncoprotein contains sequences that are essential for cellular transformation (S. Farina, et al. J. Virol., 66: 2698-2708, 1992; S. Min and E. J. Taparowsky. Oncogene, 7:1531-1540, 1992) and for the ability to activate gene transcription (S. Min and E. J. Taparowsky. Oncogene, 7:1531-1540, 1992). To investigate the molecular interactions that mediate these v-Myc-associated activities, we performed competition assays in which various regions of the v-Myc amino terminal transcription activation domain (TAD) were examined for their ability to inhibit transcription activation by v-Myc, VP16, and the myogenic regulatory factor MyoD. Overexpression of these transcriptional activators also was used to investigate whether Myc-interacting proteins were required for cellular transformation and cell proliferation events. Our results demonstrate that at least two distinct cellular activities interact with the v-Myc TAD and that it is the synergism between these activities that is required for v-Myc to function fully as a transcriptional activator. In addition, v-Myc activators squelch VP16- and MyoD-dependent transcription activation, suggesting that the v-Myc TAD interacts with a component of the general transcription machinery. In support of this observation, we found that overexpression of the v-Myc TAD inhibits ras-mediated cellular transformation as well as cell proliferation, underscoring the critical role these amino terminal Myc-interacting factors play in regulating the physiology of both normal and transformed cells.

Overproduction of v-Myc in the nucleus and its excess over Max are not required for avian fibroblast transformation.

The cellular proto-oncogene c-myc can acquire transforming potential by a number of different means, including retroviral transduction. The transduced allele generally contains point mutations relative to c-myc and is overexpressed in infected cells, usually as a v-Gag-Myc fusion protein. Upon synthesis, v-Gag-Myc enters the nucleus, forms complexes with its heterodimeric partner Max, and in this complex binds to DNA in a sequence-specific manner. To delineate the role for each of these events in fibroblast transformation, we introduced several mutations into the myc gene of the avian retrovirus MC29. We observed that Gag-Myc with a mutated nuclear localization signal is confined predominantly in the cytoplasm and only about 5% of the protein could be detected in the nucleus (less than the amount of endogenous c-Myc). Consequently, only a small fraction of Max is associated with Myc. However, cells infected with this mutant exhibit a completely transformed phenotype in vitro, suggesting that production of enough v-Gag-Myc to tie up all cellular Max is not needed for transformation. While the nuclear localization signal is dispensable for transformation, minimal changes in the v-Gag-Myc DNA-binding domain completely abolish its transforming potential, consistent with a role of Myc as a transcriptional regulator. One of its potential targets might be the endogenous c-myc, which is repressed in wild-type MC29-infected cells. Our experiments with MC29 mutants demonstrate that c-myc down-regulation depends on the integrity of the v-Myc DNA-binding domain and occurs at the RNA level. Hence, it is conceivable that v-Gag-Myc, either directly or circuitously, regulates c-myc transcription.

EGF receptor activity and mitogenic response of Balb/3T3 cells expressing Ras and Myc oncogenes. EGF receptor activity in oncogene transformed cells.

EGF receptors are found on the surface of most cells, usually with high and low binding affinities. To investigate functional relationships between EGF (EGF-like growth factors) and oncogenes we have characterized the expression of the epidermal growth factor receptor (EGFr) in H-Ras, v-Myc, and H-Ras-v-Myc transformed Balb/3T3 cells. H-Ras cells show a marked decrease in the number of EGFr molecules per cell compared to parental cells. v-Myc and H-Ras-v-Myc transformed cells express an intermediate level of receptors. The majority of the EGF receptors on the parental and oncogene transformed cells bind EGF with low affinity and this low affinity receptor is down-regulated by oncogene transformation. v-Myc expression, in the H-Ras-v-Myc transformed cells, abrogates the receptor down-regulation seen with H-Ras transformation. The mechanism of abrogation is not a result of a change in the p21-Ras concentration in the H-Ras-v-Myc transformed cells. In addition, the mitogenic response to EGF was examined. H-Ras and H-Ras-v-Myc transformed cells do not respond to EGF mitogenically. In contrast, EGF stimulates DNA synthesis in parental cells and v-Myc transfected cells; this result suggests that growth promoting signals from the EGF receptor may not be required in H-Ras transformed cells.

Mutations within the 5' half of the avian retrovirus MC29 v-myc gene alter or abolish transformation of chicken embryo fibroblasts and macrophages.

Avian myelocytomatosis virus MC29 induces a wide variety of neoplastic diseases in infected birds and transforms cells of the macrophage lineage as well as fibroblasts and epithelial cells. A biological and biochemical analysis, carried out on a series of in-frame insertion and deletion mutations within the gag-myc gene of MC29, revealed several mutations within the 5' portion of the v-myc gene that encode proteins either completely defective for transformation or compromised in their ability to transform chicken embryo fibroblasts but not macrophages. Mutations within the 3' end of the v-myc gene which disrupt sequences encoding the basic/helix-loop-helix region were defective for transformation of both fibroblasts and macrophages. Eight variants were cloned into the replication-competent avian expression vector RCAS. Analysis of cells infected with transformation-defective, replication-competent viruses confirmed the expression of functionally defective Myc proteins. Further, expression of the transformation defective variant dl91-137 in chicken fibroblasts inhibited subsequent transformation by wild-type MC29. The results reported herein support the hypothesis that Myc proteins function as regulators of transcription in a variety of cell types and clearly point out the necessity of putative regulatory domains within the amino-terminal half of the Myc protein.

In vitro modulation and relationship between N-myc and HLA class I RNA steady-state levels in human neuroblastoma cells.

Neuroblastoma cell lines and tumors are characterized by low HLA class I expression. The majority of neuroblastoma cell lines and a high percentage of disseminated tumors display amplification of the nuclear protooncogene N-myc. An inverse correlation between HLA class I expression and N-myc amplification and overexpression has been recently described in neuroblastomas (NBs). In this study we have shown that cytokines (recombinant gamma-interferon, recombinant alpha-tumor necrosis factor), differentiation agents (dibutyryl cyclic AMP, phorbol myristate acetate) and growth factors (nerve growth factor, epithelial growth factor) were able to influence the growth rate and surface expression of HLA class I molecules as well as of a tumor-associated antigen on 2 representative NB cell lines. Induced decreased growth rate in NB cells was not always related to decreased N-myc expression. Analysis at the mRNA level revealed that both N-myc and HLA class I RNA steady-state levels could be modulated by several substances, including recombinant gamma-interferon, phorbol myristate acetate, dibutyryl cyclic AMP, and epithelial growth factor and were not necessarily linked. An inverse correlation between N-myc and HLA mRNA levels was observed only after exposure of NB cells to recombinant alpha-tumor necrosis factor. We conclude that N-myc and HLA class I RNA steady-state levels can be modulated independently and suggest that they are not necessarily inversely regulated.