ALBERT

Description: Multiple myeloma (MM), a clonal B-cell malignancy, characterized by accumulation of plasma cells in bone marrow is the second most hematological malignancy in United States. In the BM, the myeloma and stromal cells secrete cytokines, which support the growth and survival of the myeloma cells. We previously showed that JAG2, one of the NOTCH ligand, is over-expressed in MM cells (Blood. 2004 Dec 1;104(12)). We hypothesized that over-expression of JAG2 in myeloma cells induce secretion of IL-6 from the stromal cells and subsequently enhances the proliferation of myeloma cells. JAG2 has been shown to be over-expressed in all cell lines and patient samples studied To identify the mechanism for JAG2 overexpression in MM cells, we assessed the potential modifications of the JAG2 promoter in MM cell lines as well as patient samples (an JAG2 negative controls) by studying both methylation and histone acetylation level of the JAG2 promoter. We show that difference in H4 acetylation level might play a crucial role in JAG2 expression. Acetylation state of histones can be regulated by the recruitment of histone deacetylases (HDACs). HDACs are typically recruited to promoter regions through interaction with nuclear co-repressors such as SMRT. The cell lines and patient samples studied presented significantly reduced levels of SMRT. Therefore, based on these observations we propose a model in which partial down-regulation of SMRT recruits less active HDAC3 (as confirmed by immunoprecipitation). As a result the deactylation process of histones in the JAG2 promoter region has been impaired and the cells lost their regulatory mechanism on transcriptional regulation of JAG2. This provides a mechanistic explanation for JAG2 over-expression in MM and on the direct involvement of the SMRT co-repressor in MM pathogenesis.

Description: The Silencing Mediator of retinoid and Thyroid hormone (SMRT) is a transcription co-repressor whose association with nuclear receptors both in solution and bound to DNA response elements is destabilized by ligand. SMRT and a related co-repressor N-CoR are recruiting a transcriptional repression complex, that contains sin3A/B protein and histone deacetylases (HDAC1/2) to nuclear receptors. It is now well established that SMRT can interact with non-receptor proteins such as BCL6, for its repression activities. The ability of the “silencing complex” to deacetylate histones results in a condensed chromatin state that is inhibitory to transcription. We have previously shown that SMRT was localized at chromosome 12q24. In addition, down-regulation of the SMRT protein due to 12q24 rearrangements was recurrently associated with NHL transformation, supporting a tumor suppressor function for SMRT (Cancer Res, 65(11):4554–4561, 2005). We further investigated the reasons why the 12q24 region and SMRT was targeted by chromosomal rearrangements. Genetic breakage is one mechanism by which functional loss of tumor suppressor gene activity may occur. Chromosomal locations in which genetic breakage may be induced are known as fragile sites. Fragile sites have been shown to be involved in some malignancies in which the fragile site lies within known genes, such as the FHIT gene (chromosome 3p) in lung cancer, and where small deletions are consistently observed on chromosome 3. Two fragile sites exist on the long arm of chromosome 12. FRA12B is located at 12q24.13 and FRA12E has been located at 12q24.2–3. Interestingly the FRA12E region corresponds to the site of SMRT (12q24.2). To assess whether the FRA12E fragile site is localized within the SMRT gene, we studied normal lymphocytes cultured in the presence of aphidicolin (an inducer of fragile sites) and metaphase chromosomes were subsequently prepared following classical cytogenetics protocols. These preparations were then subjected to fluorescence in situ hybridization (FISH) using a set of SMRT-specific RPCI BAC clones. Using this approach, we were able to demonstrate that the FRA12E fragile site is localized within the SMRT gene. We further characterize several lymphoblastoid cell lines that were either carrier or not of the FRA12E fragile site. This provides us with a mechanistic explanation for recurrent 12q24 rearrangements seen in transformed NHLs. The FRA12E-carrier lymphoblastoid cell lines will provide us the opportunity to characterize the structure of this fragile site.

Description: Leukemia is a neoplastic proliferation of cells of hematopoietic origin that arises following somatic mutation in a single hematopoietic stem cell, the progeny of which forms a clone of leukemia cells. Genetic alterations leading to leukemia transformation of a cell are often associated with major alterations of chromosomes that can be detected by studying cells of the leukemic clone in mitosis. One of these alterations is a chromosomal translocation that is often used to identify genes potentially involved in other type of rearrangements such as deletions. Abnormalities of chromosome band 13q14 occur in hematologic malignancies of all lineages and at all stages of differentiation. Unlike other chromosomal translocations, which are usually specific for a given lineage, the chromosomal translocation t(12;13)(p12;q14) has been observed in both B-cell and T-cell precursor acute lymphoblastic leukemia (ALL), in differentiated and undifferentiated acute myeloid leukemia (AML) and in chronic myeloid leukemia at progression to blast crisis (CML-BC). Recently, we have shown the presence of a myeloid- and lymphoid-specific breakpoint cluster regions within chromosome band 13q14 in acute leukemia (Genes Chromosome Cancer 25:222-229,1999). In addition, a new cell line has been established from one of the lymphoid cases, MUTZ5, that carries a single t(12;13) translocation (Leukemia15:1471-1474, 2001). The molecular characterization of this translocation led to the identification of a new gene, FLJ13639, that is disrupted and lost in the MUTZ5 cell line. This gene shares homologies with the large family of short-chain dehydrogenase reductase (SDR). Furthermore, three transcripts and proteins were found to be differentially expressed for this gene, where P1 is potentially the active form of dehydrogenase, while P2 and P3 are lacking the co-activator site. We previously reported that one of the consequences of the loss of FLJ13639 is the over-expression of CD24 that appears to provide leukemia cells with a proliferation and invasiveness advantage, as well as a certain degree of chemoresistance. In addition, data on patient samples indicated a correlation between survival and CD24High/FLJ/P1Low expression profile (Blood 106 (11), Nov 2005). We show that FLJ/P1 is a new mitochondrial protein that is important for the respiration and apoptosis processes. Restoration of the FLJ/P1 function induced CD24 down-regulation, decreased invasive potential as assessed by Matrigel assay and decreased chemoresistance. FLJ/P1 function restoration might represent a new potential therapy, that could, when combined with the assessment of FLJ/P1-CD234 expression profiles, allow the future development of personalized treatment.

Description: Radioimmunotherapy with anti-CD20 monoclonal antibodies is a promising new treatment approach for patients with relapsed B-cell lymphomas. However, the majority of patients treated with conventional radiolabeled anti-CD20 antibodies eventually have a relapse because the low tumor-to-blood and tumor-to–normal organ ratios of absorbed radioactivity limit the dose that can be safely administered without hematopoietic stem cell support. This study assessed the ability of a streptavidin-biotin “pretargeting” approach to improve the biodistribution of radioactivity in mice bearing Ramos lymphoma xenografts. A pretargeted streptavidin-conjugated anti-CD20 1F5 antibody was infused, followed 24 hours later by a biotinylated N-acetylgalactosamine–containing “clearing agent” and finally 3 hours later by 111In-labeled DOTA-biotin. Tumor-to-blood ratios were 3:1 or more with pretargeting, compared with 0.5:1 or less with conventional 111In-1F5. Tumor-to–normal organ ratios of absorbed radioactivity up to 56:1 were observed with pretargeting, but were 6:1 or less with conventional 111In-1F5. Therapy experiments demonstrated that 400 μCi (14.8 MBq) or more of conventional 90Y-1F5 was required to obtain major tumor responses, but this dose was associated with lethal toxicity in 100% of mice. In marked contrast, up to 800 μCi (29.6 MBq)90Y-DOTA-biotin could be safely administered by the pretargeting approach with only minor toxicity, and 89% of the mice were cured. These data suggest that anti-CD20 pretargeting shows great promise for improving current therapeutic options for B-cell lymphomas and warrants further preclinical and clinical testing.