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In human follicular lymphoma the t(14; 18) chromosome translocation activates the antiapoptotic oncogene Bcl2 by linking it to the immunoglobulin heavy chain (IGH) locus. Transgenic mice expressing Bcl2 controlled by an Igh enhancer (Eμ) do not develop follicular lymphoma, although they do have an increased incidence of other B-lymphoid neoplasms. We have now analyzed tumorigenesis in mice bearing a Bcl2 transgene controlled by Vav gene regulatory sequences (VavP), which confer expression in multiple hematopoietic lineages. Unlike Eμ-Bcl2 mice, many VavP-Bcl2 mice older than 10 months developed follicular lymphoma. Young VavP-Bcl2 mice had an overabundance of enlarged germinal centers and greatly elevated numbers of cycling B cells that had undergone IgH class switching and V-gene hypermutation. The peripheral T-cell compartment was larger in the VavP-Bcl2 mice than in Eμ-Bcl2 strains and, notably, CD4 T cells were 5-fold increased over normal. The germinal center hyperplasia required CD4 T cells, because it could be abolished by anti-CD4 antibody in vivo. VavP-Bcl2 mice also had a propensity to develop kidney disease of the autoimmune type. We suggest that the increased survival capacity of B and T cells fosters prolonged germinal center reactions, and that autoreactivity and hypermutation conspire to generate follicular lymphoma.

Over-expression of the c-MYC oncogene and Trp53 gene mutations are among the most common genetic alterations in human cancer and, when combined, result in highly aggressive malignant disease. Trp53 gene mutations produce over-expressed mutant TRP53 proteins that drive cancer growth through both loss of wild-type Trp53 tumor suppressor function and gain-of-function oncogenic properties. The Eμ-Myc mouse model provides a setting to study the functional interplay between c-Myc over-expression and mutant TRP53 proteins. Eμ-Myc transgenic mice carry a c-Myc transgene under the control of the immunoglobulin heavy chain gene enhancer (Eμ), recapitulating the chromosomal translocation underlying human Burkitt Lymphoma, and develop aggressive pre-B/B cell lymphoma with a high (~20%) spontaneous rate of Trp53 mutation. The effect of five mouse mutant TRP53 proteins (V170M, I192S, G280, R246Q, R270H) was initially examined in three settings (Trp53-/-, Trp53+/- and Trp53+/+;Eμ-Myc) using a hematopoietic stem and progenitor cell (HSPC) reconstitution model. Each mutant TRP53 protein studied corresponds to a commonly re-occurring Tp53 mutation in human cancer. Retroviral over-expression enabled the comparison of mutant-specific and genotype-specific features for each mutant TRP53 protein. Mutant TRP53 expression did not accelerate lymphoma development in mice receiving Trp53-/- or Trp53+/- HSPCs. However, mice reconstituted with Trp53+/- HSPCs expressing the TRP53 mutants displayed an altered tumor spectrum compared to mice reconstituted with control Trp53+/- HSPCs. In contrast, mutant TRP53 markedly accelerated lymphoma development in mice receiving Trp53+/+;Eμ-Myc HSPCs, highlighting a synergy between c-Myc over-expression and Trp53 mutations in neoplastic transformation. Furthermore, inducible mutant TRP53 expression demonstrated a dependency on sustained expression of mutant TRP53 in established MYC-driven lymphomas. Notably, none of the c-MYC plus mutant TRP53 driven lymphomas exhibited spontaneous endogenous Trp53 mutations. Despite the enhanced tumorigenesis, most established lymphomas from this model displayed sensitivity to TRP53-activating drugs consistent with a weak dominant negative effect over wild-type Trp53-induced apoptosis. Consistent with this finding, pre-malignant Trp53+/+;Eμ-Myc primary B-cells expressing mutant TRP53 were not protected against Trp53-induced apoptosis. Pre-malignant B-cells displayed a small increase in cell cycling and an expansion of the tumor-initiating pre/pro-B cell population. Most significantly, functional assessment of DNA damage in pre-malignant cells, using single cell gel electrophoresis (comet assay) and γ-H2AX staining, revealed increased DNA damage, suggesting an important role for defects in DNA repair during mutant TRP53-driven lymphoma development. To investigate the nature of the dominant negative effect, mutant TRP53 protein was exogenously expressed in mouse Eµ-Myc Trp53+/+ lymphoma cell lines. The impact of mutant TRP53 on the transcriptional function of the endogenous wild-type TRP53 protein was then studied using the TRP53-activating compound, nutlin-3a. Surprisingly, in established lymphoma cell lines, mutant TRP53 impaired nutlin-3a-induced apoptosis despite substantial induction of the critical pro-apoptotic effector, PUMA. To explore this finding further, we globally characterized the dominant negative effect, and assessed for mutant TRP53-specific transcriptional targets, by performing whole transcriptome sequence (RNAseq) analysis after treatment with nutlin-3a. Analysis of known wild-type Trp53 target genes (n=283) demonstrated that the induction of these genes as a group was repressed in the presence of the mutant TRP53 protein (ROAST test, p=6.7e-04). Remarkably, however, mutant TRP53 significantly repressed only 57% of the nutlin-3a-induced Trp53 target genes. Analysis of these strongly repressed genes highlighted the importance of several pathways, including metabolism, DNA damage repair and negative feedback loops in TRP53 signaling. This suggests a previously unrecognized selectivity of the dominant-negative-effect for certain p53 pathways that may be important in cancer initiation. Additional mutant TRP53-specific transcriptional targets were also identified and are under further investigation. Disclosures No relevant conflicts of interest to declare.

Abstract 389 The survival of megakaryocytes and platelets is regulated by the intrinsic apoptosis pathway. Both cell types express Bak and Bax, the essential mediators of intrinsic apoptosis, which must be kept in check for cellular viability to be maintained. In platelets, Bak and Bax are restrained by the pro-survival protein Bcl-xL. Mutations that reduce the pro-survival activity of Bcl-xL cause dose-dependent cell-intrinsic reductions in circulating platelet life span in mice. Accordingly, pharmacological blockade of Bcl-xL with the BH3 mimetic drugs ABT-737 or ABT-263 (Navitoclax) triggers platelet death and thrombocytopenia in mice, dogs and humans. In mice, loss of Bak and Bax almost doubles platelet life span, and renders platelets refractory to the effects of ABT-737. In megakaryocytes, we and others have recently demonstrated that Bcl-xLand its pro-survival relative Mcl-1 are essential for restraint of the intrinsic apoptosis pathway. Their loss triggers Bak/Bax-mediated death. Conversely, ablation of Bak and Bax can protect megakaryocytes from acute apoptotic insults, such as treatment with carboplatin. Combined with the fact that platelet production is normal in the absence of Bak and Bax, these studies have brought into question the long-standing theory that megakaryocytes deliberately undergo apoptosis in order to shed platelets. However, whilst it is clear that the intrinsic apoptosis pathway is not required for thrombopoiesis, the role of the extrinsic pathway—the other major route to apoptotic cell death—has not been established. In the current study we examined the functionality of, and physiological requirement for, the extrinsic apoptosis pathway in megakaryocytes and platelets. The extrinsic pathway is triggered when members of the tumor necrosis factor (TNF) superfamily such as Fas ligand (FasL) bind to cell surface death receptors (e.g. Fas). This induces receptor multimerization, recruitment of death domain adaptor proteins (e.g. FADD) and subsequent activation of Caspase-8, which is the essential mediator of extrinsic pathway-mediated cell death. We found that both megakaryocytes and platelets express critical components of the pathway, including FADD, Caspase-8 and Bid. Megakaryocytes, but not platelets, also expressed the death receptor Fas. Mature fetal liver-derived megakaryocytes treated with soluble FasL exhibited activation of Caspase-8 and the effector Caspases-3/7. This was accompanied by mitochondrial damage and a failure of pro-platelet formation. To establish the requirement for the extrinsic pathway in megakaryocyte development and platelet production, we conditionally deleted Caspase-8 from the megakaryocyte lineage. Platelet counts and platelet life span in Casp8Pf4Δ/Pf4Δ mice were indistinguishable from those of wild-type littermates. Megakaryocyte numbers, morphology, ploidy and in vitro pro-platelet formation capacity were also normal. Caspase-8-deficient megakaryocytes were resistant to FasL treatment. Casp8Pf4Δ/Pf4Δ animals responded to experimentally-induced thrombocytopenia in a manner similar to wild-type. Collectively, these data indicate that the extrinsic apoptosis pathway is dispensable for the generation and survival of platelets. To examine any potential redundancy between the extrinsic and intrinsic apoptosis pathways, we generated Bak−/−BaxPf4Δ/Pf4ΔCasp8Pf4Δ/Pf4Δ triple knockout mice. The ability of these animals to produce platelets, both at steady state and under conditions of thrombopoietic stress, was unperturbed. Megakaryocyte numbers and morphology were normal. Thus, platelet shedding by megakaryocytes does not require the intrinsic or extrinsic apoptosis pathways. Together with recent work demonstrating that the apoptotic initiator caspase, Caspase-9, is also dispensable for platelet production, we conclude that platelet biogenesis is not an apoptotic process. Disclosures: No relevant conflicts of interest to declare.