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Both the canonical and noncanonical nuclear factor κB (NF-κB) pathways have been linked to tumorigenesis. However, it remains unknown whether and how the 2 signaling pathways cooperate during tumorigenesis. We report that inhibition of the noncanonical NF-κB pathway significantly delays tumorigenesis mediated by the viral oncoprotein Tax. One function of noncanonical NF-κB activation was to repress expression of the WWOX tumor suppressor gene. Notably, WWOX specifically inhibited Tax-induced activation of the canonical, but not the noncanonical NF-κB pathway. Mechanistic studies indicated that WWOX blocked Tax-induced inhibitors of κB kinaseα (IKKα) recruitment to RelA and subsequent RelA phosphorylation at S536. In contrast, WWOX Y33R, a mutant unable to block the IKKα recruitment and RelA phosphorylation, lost the ability to inhibit Tax-mediated tumorigenesis. These data provide one important mechanism by which Tax coordinates the 2 NF-κB pathways for tumorigenesis. These data also suggest a novel role of WWOX in NF-κB regulation and viral tumorigenesis.

Infection with the human T-cell leukemia virus-1 (HTLV-1) results in a variety of diseases including adult T-cell leukemia/lymphoma (ATL). Although the pathogenesis of these disorders is poorly understood, it involves complex interactions with the host immune system. Activation of infected T cells may play an important role in disease pathogenesis through induction of the oncogenic HTLV-1 Tax transactivator protein. To test this hypothesis, we employed transgenic mice in which Tax is regulated by the HTLV-1 LTR. T-cell receptor stimulation of LTR-Tax CD4+ T cells induced Tax expression, hyper-proliferation, and immortalization in culture. The transition to cellular immortalization was accompanied by markedly increased expression of the antiapoptotic gene, mcl-1, previously implicated as important in T-cell survival. Immortalized cells exhibited a CD4+CD25+CD3− phenotype commonly observed in ATL. Engraftment of activated LTR-Tax CD4+ T cells into NOD/Shi-scid/IL-2Rγ null mice resulted in a leukemia-like phenotype with expansion and tissue infiltration of Tax+, CD4+ lymphocytes. We suggest that immune activation of infected CD4+ T cells plays an important role in the induction of Tax expression, T-cell proliferation, and pathogenesis of ATL in HTLV-1–infected individuals.

Abstract 258 PDCD2 (Programmed cell death domain 2) is a highly conserved zinc finger MYND domain-containing nuclear protein expressed in a variety of tissues. Our group has demonstrated that in Drosophila, PDCD2 mutants display massive lymph gland hyperplasia phenotype suggestive of hematopoietic hyperproliferation concomitant with a block in differentiation. The human PDCD2 gene is located on chromosome 6q27 and encodes two alternative transcripts with multiple splice variants. We analyzed primary human leukemias and multiple tumor cell lines and detected a PDCD2 splice variant whose expression is associated exclusively with malignant but not normal hematopoietic samples. To study the role of alternative PDCD2 splicing in hematopoiesis in a biologically relevant vertebrate, we isolated the zebrafish PDCD2 and found that it is expressed in early embryonic stages, and its expression latter on colocalized with the hemangioblast and hematopoietic stem cell (HSC) markers cMyb, Flk1 and RUNX1. Promoter analysis demonstrated multiple binding sites for hematopoietic and lymphoid transcription factors controlling embryonic and adult hematopoiesis. We next generated transgenic zebrafish expressing EGFP under the control of the PDCD2 promoter. EGFP expression in zebrafish recapitulates PDCD2 expression pattern in HSC. Morpholino-mediated PDCD2 knockdown resulted in developmental defects with marked decrease in the number of embryonic blood cells and circulatory failure at 4 dpf. Morpholino studies using transgenic zebrafish with labeled endothelial and HSC progenitors demonstrated that the effects of PDCD2 knockdown are restricted to HSC but not endothelial progenitors. Concordant with this observation, we examined the expression of several markers for early hematopoiesis including SCL, RUNX1 and cMyb using in situ hybridization (ISH). PDCD2 knockdown resulted in a severe reduction in RUNX1+ cells suggesting that HSC formation in these embryos is compromised. Our ISH analysis was extended to examine the effects of PDCD2 loss on lineage-committed hematopoiesis with Gata1, Mpx, L-plastin, Ikaros, Tcr-α, Lck, and IgM hematopoietic lineage-specific probes. Utilizing a competitive RT-PCR assay, we found that in zebrafish leukemias, similar to human leukemias, a unique (tumor–specific) PDCD2 isoform is upregulated. To investigate the tumor suppressor and/or oncogenic function of these PDCD2 isoforms, we have generated multiple transgenic zebrafish expressing the different PDCD2 splice variants. Overexpression of PDCD2 variants derived from the alternatively spliced PDCD2 mRNAs resulted in an overt leukemic phenotype in 30% of the transgenic fish. Preliminary examination of the affected fish suggests that PDCD2 overexpression has the potential to drive leukemogenesis in either the myeloid, lymphoid, or erythroid lineages in individual cases. Moreover, we observed a bias toward erythroleukemia (M6) in the case of one isoform. Our data demonstrate a spectrum of leukemia arising from PDCD2 dysregulation. In light of our analysis of the normal PDCD2 function in hematopoiesis, we conclude that the alternative isoforms of PDCD2 play an important role in regulating normal HSC functions, and deviation from this role is a powerful promoter of leukemogenesis. Disclosures: Steward: UMDNJ: Patents & Royalties. Schaar:UMDNJ: Patents & Royalties. Sabaawy:UMDNJ: Patents & Royalties.