The proto-oncogene c-myc has been shown to play a pivotal role in cell cycle regulation, metabolism, apoptosis, dierentiation, cell adhesion, and tumorigenesis, and participates in regulating hematopoietic homeostasis. It is a transcription regulator that is part of an extensive network of interacting factors. Most probably, dierent biological responses are elicited by dierent overlapping subsets of c-Myc target genes, both induced and suppressed. Results obtained from studies employing mouse models are consistent with the need for at least one, and possibly two, mutations in addition to deregulated c-myc for malignant tumor formation. Repression of c-myc is required for terminal dierentiation of many cell types, including hematopoietic cells. It has been shown that deregulated expression of c-myc in both M1 myeloid leukemic cells and normal myeloid cells derived from murine bone marrow, not only blocked terminal dierentiation and its associated growth arrest, but also induced apoptosis, which is dependent on the Fas/CD95 pathway. There is evidence to suggest that the CD95/Fas death receptor pathway is an integral part of the apoptotic response associated with the end of the normal terminal myeloid dierentiation program, and that deregulated c-myc expression can activate this signaling pathway prematurely. The ability of egr-1 to promote terminal myeloid dierentiation when coexpressed with c-myc, and of c-fos to partially abrogate the block imparted by deregulated c-myc on myeloid dierentiation, make these two genes candidate tumor suppressors. Several dierent transcription factors have been implicated in the down-regulation of c-myc expression during dierentiation, including C/EBPa, CTCF, BLIMP-1, and RFX1. Alterations in the expression and/or function of these transcription factors, or of the c-Myc and Max interacting proteins, such as MM-1 and Mxi1, can in¯uence the neoplastic process. Understanding how c-Myc controls cellular phenotypes, including the leukemic phenotype, should provide novel tools for designing drugs to promote dierentiation and/ or apoptosis of leukemic cells.

Both deregulated growth and blocks in differentiation cooperate in the multistage process of leukemogenesis. Thus, understanding functional interactions between genes that regulate normal blood cell development, including cell growth and differentiation, and how their altered expression contributes to leukemia, is important for rational drug design. Previously, we have shown that the zinc finger transcription factor Egr-1 plays a role in monocytic differentiation.

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Mutated p53 genes are capable of complementing activated ras genes in the transformation of primary rat embryo fibroblasts in vitro. Mutations in both genes have also been found in several human cancers, including lung carcinomas. We generated transgenic mice containing a p53 construct with a missense mutation in exon 5 (ala135val) to study the role of p53 mutations in lung tumorigenesis, and to facilitate identification of other genetic events that might complement p53 mutations in mouse lung carcinogenesis. The p53 transgenic lines exhibited a higher frequency of lethal lung tumors than the parental FVB/N strain. We examined the spontaneously-arising lung carcinomas from mice expressing the mutated p53 transgene for K-ras mutations using single-stranded conformation polymorphism (SSCP) and/or direct sequencing approaches. Fifteen of 29 (52%) carcinomas contained mutations in the K-ras oncogene. Six of 15 of the K-ras mutations were in codon 61 and 9/15 were in codon 12. Subsequent analysis of spontaneous lung carcinomas from mice of the FVB/N parental strain showed that 9/12 (75%) carcinomas examined contained K-ras mutations. Two of these were in codon 12, one in codon 13, and 6 were in codon 61. These results demonstrate that the frequency of ras mutations does not differ between the p53 FVB/N transgenic mice and their parental FVB/N strain but suggest that a high frequency of mutations K-ras can be correlated with lung tumorigenesis in both groups of mice.

3723 Poster Board III-659 Introduction Rituximab-containing chemoimmunotherapy regimens have become the standard of care for first line therapy of follicular lymphoma (FL). Despite such intensive treatments most FL patients will continue to relapse and prognosis for those not cured with initial therapy remains poor. Lenalidomide is an immunomodulatory agent; in several in vitro and ex vivo lymphoma models lenalidomide has shown an ability to induce both growth arrest and cell kill via both immune-mediated and direct cytotoxicity. A recent clinical study in relapsed/refractory FL heavily pretreated patients achieved a duration of response lasting >16.5 months in response to lenalidomide treatment (Witzig et al JCO in press). Preliminary results from smaller studies in FL also indicate that combining lenalidomide with rituximab (R2) can achieve remarkable responses in the newly diagnosed and in the relapsed/refractory settings. Here we aim to better characterize the non-immune mediated activity of lenalidomide in NHL cell lines and determine if lenalidomide-mediated cytotoxicity is affected by the presence of rituximab. Methods NHL cell lines used in the study included DoHH-2 (FL), Rec-1 (Mantle Cell Lymphoma) and Farage (Diffuse Large B cell Lymphoma). The range in concentrations of lenalidomide and rituximab was from 0.0001 to 100 μM, and from 0.0001 to 100 mg/ml, respectively, depending on the individual sensitivity of each cell line. In vitro tumor cell proliferation assays were done by incubating each cell line with each drug separately and in combination for 72 hrs, and measuring proliferative activity by 3H-thymidine incorporation. A proliferation inhibition index for each agent alone and combined was calculated via the Chou Talalay method (CompuSyn software) and expected additive effects were calculated using the fractional product method. Apoptosis was analyzed via Annexin V and propidium iodide (PI) staining and quantified using flow cytometry, which was also used to measure CD20 expression. Phospho-Bcl2 levels in drug-treated DoHH2 cells were assessed via western blot. Results Lenalidomide induced approx. 40% and 20% inhibition in proliferative activity of both DoHH2 and Rec-1 cells, respectively. Rituximab inhibited the proliferation of DoHH2 and Rec-1 cells by approx. 50% and 20%, respectively, and additive antiproliferative activity (40%) resulted from treatment of Rec-1 cells with R2. The Farage DLBCL cell line showed approx. 25% inhibition in proliferation with lenalidomide and rituximab individually and additive anti-proliferative activity when treated with R2. However, synergistic inhibition in proliferation (85% inhibition) was achieved when DoHH2 cells were treated with R2. Further, lenalidomide did not affect surface CD20 expression of any cells examined. R2 inhibited proliferation of DoHH2 cells at combination index values ranging from 0.0045 to 0.023, and directly potentiated DoHH2 cells to undergo apoptosis. Synergy in apoptotic activity was also demonstrated ex vivo in cells from a FL patient treated with R2. In DoHH2 cells, addition of lenalidomide was found to potentiate the rituximab-mediated phosphorylation of Bcl-2, thus inactivating its activity and promoting a loss in mitochondrial membrane potential that results in release of cytochrome C to trigger caspase-mediated apoptosis. Conclusions These results indicate that R2 induces synergistic anti-proliferative and pro-apoptotic effects in both FL cell line and in primary cells. Evidence suggests that this direct cytotoxicity is mediated by signaling events involving Bcl-2 activation leading to cell death. These data support clinical findings showing a benefit in combining lenalidomide with rituximab and warrant further investigation of R2 in future lymphoma trials. Disclosures: Gandhi: Celgene: Employment, Equity Ownership. Kang:Celgene: Employment. Capone:Celgene: Employment. Shafarenko:Celgene: Employment. Schafer:Celgene: Employment.