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Histone deacetylases (HDACs) are epigenetic erasers of lysine-acetyl marks. Inhibition of HDACs using small molecule inhibitors (HDACi) is a potential strategy in the treatment of various diseases and is approved for treating hematological malignancies. Harnessing the therapeutic potential of HDACi requires knowledge of HDAC-function in vivo. Here, we generated a thymocyte-specific gradient of HDAC-activity using compound conditional knockout mice for Hdac1 and Hdac2. Unexpectedly, gradual loss of HDAC-activity engendered a dosage-dependent accumulation of immature thymocytes and correlated with the incidence and latency of monoclonal lymphoblastic thymic lymphomas. Strikingly, complete ablation of Hdac1 and Hdac2 abrogated lymphomagenesis due to a block in early thymic development. Genomic, biochemical and functional analyses of pre-leukemic thymocytes and tumors revealed a critical role for Hdac1/Hdac2-governed HDAC-activity in regulating a p53-dependent barrier to constrain Myc-overexpressing thymocytes from progressing into lymphomas by regulating Myc-collaborating genes. One Myc-collaborating and p53-suppressing gene, Jdp2, was derepressed in an Hdac1/2-dependent manner and critical for the survival of Jdp2-overexpressing lymphoma cells. Although reduced HDAC-activity facilitates oncogenic transformation in normal cells, resulting tumor cells remain highly dependent on HDAC-activity, indicating that a critical level of Hdac1 and Hdac2 governed HDAC-activity is required for tumor maintenance.

Histone deacetylases (HDACs) are epigenetic erasers of lysine-acetyl marks. Inhibition of HDACs using small molecule inhibitors (HDACi) is a potential strategy in the treatment of various diseases and is approved for treating hematological malignancies. Harnessing the therapeutic potential of HDACi requires knowledge of HDAC-function in vivo. Here, we generated a thymocyte-specific gradient of HDAC-activity using compound conditional knockout mice for Hdac1 and Hdac2. Unexpectedly, gradual loss of HDAC-activity engendered a dosage-dependent accumulation of immature thymocytes and correlated with the incidence and latency of monoclonal lymphoblastic thymic lymphomas. Strikingly, complete ablation of Hdac1 and Hdac2 abrogated lymphomagenesis due to a block in early thymic development. Genomic, biochemical and functional analyses of pre-leukemic thymocytes and tumors revealed a critical role for Hdac1/Hdac2-governed HDAC-activity in regulating a p53-dependent barrier to constrain Myc-overexpressing thymocytes from progressing into lymphomas by regulating Myc-collaborating genes. One Myc-collaborating and p53-suppressing gene, Jdp2, was derepressed in an Hdac1/2-dependent manner and critical for the survival of Jdp2-overexpressing lymphoma cells. Although reduced HDAC-activity facilitates oncogenic transformation in normal cells, resulting tumor cells remain highly dependent on HDAC-activity, indicating that a critical level of Hdac1 and Hdac2 governed HDAC-activity is required for tumor maintenance.

Follicular lymphoma (FL) is a disease characterized by a long clinical course marked by frequent relapses that vary in clinical aggressiveness over time. Therefore, the main dilemma at each relapse is the choice for the most effective treatment for optimal disease control and failure-free survival while at the same time avoiding overtreatment and harmful side effects. The selection for more aggressive treatment is currently based on histologic grading and clinical criteria; however, in up to 30% of all cases these methods prove to be insufficient. Using supervised classification on a training set of paired samples from patients who experienced either an indolent or aggressive disease course, a gene expression profile of 81 genes was established that could, with an accuracy of 100%, distinguish low-grade from high-grade disease. This profile accurately classified 93% of the FL samples in an independent validation set. Most important, in a third series of FL cases where histologic grading was ambiguous, precluding meaningful morphologic guidance, the 81-gene profile shows a classification accuracy of 94%. The FL stratification profile is a more reliable marker of clinical behavior than the currently used histologic grading and clinical criteria and may provide an important alternative to guide the choice of therapy in patients with FL both at presentation and at relapse. (Blood. 2005;105:301-307)

Follicular lymphoma (FL) is a disease characterized by a long clinical course marked by frequent relapses that vary in clinical aggressiveness over time. Therefore, the main dilemma at each relapse is the choice for the most effective treatment for optimal disease control and failure-free survival while at the same time avoiding overtreatment and harmful side effects. The selection for more aggressive treatment is currently based on histologic grading and clinical criteria; however, in up to 30% of all cases these methods prove to be insufficient. Using supervised classification on a training set of paired samples from patients who experienced either an indolent or aggressive disease course, a gene expression profile of 81 genes was established that could, with an accuracy of 100%, distinguish low-grade from high-grade disease. This profile accurately classified 93% of the FL samples in an independent validation set. Most important, in a third series of FL cases where histologic grading was ambiguous, precluding meaningful morphologic guidance, the 81-gene profile shows a classification accuracy of 94%. The FL stratification profile is a more reliable marker of clinical behavior than the currently used histologic grading and clinical criteria and may provide an important alternative to guide the choice of therapy in patients with FL both at presentation and at relapse. (Blood. 2005;105:301-307)

High dose chemotherapy and radiation, as applied in stem cell transplantation procedures, have been found to impair the hematopoiesis-supportive capacity of bone marrow stroma. However, the molecular mechanisms involved are far from elucidated yet. As bone marrow stromal glycosaminoglycans (GAGs) play an important role in various regulatory steps in hematopoiesis, we examined the effect of chemotherapy on the synthesis and composition of GAGs and gene expression profiles of the hematopoiesis supportive M210B4 fibroblastic stromal cell line. To this end GAGs were metabolically labeled with 3H-glucosamine and 35S-sulfate, and analyzed by conventional biochemical techniques. Cytarabine treatment resulted in a pronounced increase (1.6±0.3-fold increase) in hyaluronan (HA) and a reduction in the content (47±7% reduction) and in the extent of sulfation (3H-glucosamine to 35S-sulfate ratio increased from 2,1 versus 2,6) of heparan sulfate proteoglycans (HSPG). Gene expression analysis showed a corresponding increase in hyaluronan synthase 1 (Has 1) gene expression and a decrease in hyaluronidase 2 (Hyal 2) gene expression. The decrease in HSPG was found to correspond with downregulation of the Exostosin 1 (Ext 1) gene, encoding a bifunctional glycosyltransferase, required for the biosynthesis of HS. Finally, there was a trend towards decreased expression of the N-deacetylase/N-sulfotransferase-1 (Ndst1) gene, which might explain the decrease in sulfation of HSPG after cytarabine treatment, as deacetylation is required for sulfation of HSPG to occur. The functional consequence of the decrease in HSPG was investigated by Stromal Derived Factor-1α (SDF-1α) binding. SDF-1α was added to either untreated or treated M210B4 cells and allowed to bind for 90 minutes. After washing, bound SDF-1α was quantified by staining with the anti-SDF-1α antibody K15C. Cytarabine treatment resulted in a 31.1 ± 11.8% decline in K15C binding, indicating a decline in GAG-mediated binding of SDF-1α (n=6, Mann Whitney test, p<0.05). In conclusion, cytarabine treatment alters the expression of genes involved in GAG synthesis and degradation, thereby affecting the amount and structure of stromal GAGs. Accordingly, the chemokine-binding capacity of HSPG was negatively affected. In view of the critical role of SDF-1α in migration of stem cells and its presumed participation in the creation of the stem cell niche, our results suggest that chemotherapy-induced changes in stromal GAGs might affect homing and hematopoiesis. In addition, because of known functional capacities of HA, malignant hematopoiesis as well as tumor growth and invasion may be promoted.

High dose chemotherapy and radiation, as applied in stem cell transplantation procedures, have been found to impair the hematopoiesis-supportive capacity of bone marrow stroma. However, the molecular mechanisms involved are far from elucidated yet. As bone marrow stromal glycosaminoglycans (GAGs) play an important role in various regulatory steps in hematopoiesis, we examined the effect of chemotherapy on the synthesis and composition of GAGs and gene expression profiles of the hematopoiesis supportive M210B4 fibroblastic stromal cell line. To this end GAGs were metabolically labeled with 3H-glucosamine and 35S-sulfate, and analyzed by conventional biochemical techniques.