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Abstract 398 DNA methyltransferases (DNMT) play an important role in regulation of DNA methylation and mutations of DNMT3A are frequently found in AML. We analyzed the effects of DNMT3B overexpression on leukemogenesis using a tetracycline-inducible DNMT3B mouse model. To analyze the impact of DNMT3B overexpression on leukemogenesis, we retrovirally co- transduced lineage-negative bone marrow cells of wildtype and DNMT3B transgenic mice with an MSCV- cMyc-bcl2 or MSCV-MLL-AF9-hCD4 and an MSCV- tTA- GFP expressing vector. Using these conditions, doxycycline suppressed DNMT3B expression whereas absence of doxycycline led to overexpression of DNMT3B on the mRNA and protein level. To verify that expression of DNMT3b in this mouse model was able to induce DNA methylation, we performed Reduced Representation Bisulfite Sequencing (RRBS) on cMyc-Bcl2 leukemic bone marrow cells. Analysis of the global methylation levels revealed a strong hypermethylation in DNMT3b overexpressing bone marrow cells. Differentially methylated regions (DMR) were defined as those regions with at least 30% difference in methylation levels. DMRs were mainly located in promoter regions and first introns and the heatmap of these DMRs confirms distinct hypermethylation in DNMT3b transgenic cells whereas very few regions become hypomethylated in transgenic cells when compared to wildtype cells. This specific methylation pattern could be responsible for altered gene expression and consecutively altered (stem) cell characteristics. To analyze the influence of changed DNA methylation levels induced by expression of DNMT3b on leukemogenesis, sorted GFP+cells were transplanted into sublethally irradiated wildtype recipients. Mice with and without DNMT3B expression developed leukemia with splenomegaly and a tendency of delayed leukemogenesis in DNMT3B overexpressing mice. Mice with cMyc-Bcl2- and MLL-AF9-induced leukemia showed a clear myeloid phenotype with enhanced expression of the stem cell marker c-kit on MLL-AF9 leukemic cells. To consider the leukemogenic capacity, we transplanted the leukemic blasts into secondary recipients. cMyc-Bcl2 induced leukemia of both, wildtype and DNMT3b recipients was transplantable and lethal. DNMT3B tg leukemic cells were severely impaired in leukemia development in secondary recipients. Secondary recipients of cMyc-Bcl2 induced leukemic DNMT3B cells died significantly later (p= 0.04). As differential methylation can also have an effect on the responsiveness to chemotherapy, spleen cells from MLL-AF9 leukemic mice were treated with different cytotoxic and demethylating drugs to see if DNMT3b induced hypermethylation confers a resistance or increased sensitivity to chemotherapeutic treatment. DNMT3b overexpressing cells were more sensitive to treatment with Azacytidine and Decitabine. Taken together, these findings demonstrate that expression of DNMT3b in a doxycycline-regulatable mouse model induces widespread DNA hypermethylation, whereas a few small regions become hypomethylated when compared to wildtype cells. This specific methylation pattern is associated with delayed leukemogenesis and DNMT3b expression significantly impaired leukemia maintenance. In addition, response to chemotherapeutic treatment is altered by DNMT3b induced hypermethylation which underlines the importance of epigenetic regulatory mechanisms as promising targets for new therapeutic strategies in AML. Disclosures: Müller-Tidow: Celgene: Lecture fees Other; Deutsche Forschungsgemeinschaft: Research Funding.

Abstract 96 Acute myeloid leukemia is a clonal disease characterized by a malignant proliferation and accumulation of myeloid progenitor cells. Current therapeutic strategies are often not able to eradicate the leukemic cells. Malignancy is associated with deregulation of cell cycle check- points and the deregulation of checkpoints is associated with altered stem cell properties. A better understanding of malignant stem cells and their cell cycle regulation might help to develop new therapies. Recently, we identified p26INCA1 as a novel cell cycle regulator. GST pulldown assays revealed binding of INCA1 predominantly to CDK2- specific Cyclins and we demonstrated an inhibitory effect of INCA1 on CDK2/ CyclinA complexes in kinase activity assays. The loss of INCA1 and its inhibitory effect on the cell cycle regulation led to an increased cell cycling and consequently to an enlarged stem cell pool in vivo. Upon cytotoxic stress, the loss of Inca1 enhanced cell cycling and bone marrow exhaustion. To analyze a potential role of INCA1 in leukemogenesis we retrovirally transduced wildtype and Inca1−/− bone marrow cells with AML1-ETO9a (A1E9a) and transplanted these cells into wildtype recipients. Most of the wildtype cell- transplanted recipients died due to AML. In contrast, only one of the Inca1−/− cell- transplanted mice developed AML. Engraftment was higher upon transplantion of Inca1−/− cells but engraftment was not sustained. To consider the repopulation capacity of the leukemic cells we performed transplantation of primary leukemic cells into secondary recipients. A1E9a induced leukemia in Inca1 wildtype cells was transplantable and lethal. However Inca1−/− leukemic cells were severely impaired in leukemia development in secondary recipients. Colony forming units and replating capacity were significantly reduced in A1E9a Inca1−/− bone marrow cells although these cells showed increased CDK2 activity. Exhaustion of leukemic cells in the absence of Inca1 was confirmed by cloning efficiency assays. Further analyses were performed with c-myc induced leukemias. Interestingly, Inca1 deletion precluded the development of leukemias in secondary recipients. Taken together, these findings identify an important role for p26INCA1 in the maintenance of leukemia and potentially the self-renewal capacity of leukemic stem cells. Disclosures: No relevant conflicts of interest to declare.

Cell cycle progression is driven by the kinase activity of cyclin/CDK complexes. Dysregulation of the cell cycle leads to altered cell growth and contributes to tumorigenesis. Recently, we identified p26INCA1 as novel interaction partner of Cyclin A1/CDK2. Here, we characterize the phenotype of Inca1-null mice to uncover the cellular and molecular function of Inca1. Inca1-knockout mice were viable and fertile. FACS analyses revealed that aging mutant animals harbored an increased hematopoietic stem cell (HSC) pool. Bone marrow cells of young mice exhibited enhanced clonogenic replating efficiency in colony formation assays as compared to wildtype mice. Weekly administration of the myeloablative agent 5-fluorouracil (5-FU) led to a significantly shorter life span of Inca1−/ − mice compared to wildtype littermates. The increased 5-FU toxicity might thus be related to a higher number of cycling HSC in Inca1−/ − bone marrow. Analysis of the impact of Inca1 on cell cycle regulation demonstrated that the fraction of Inca1−/ − embryonic fibroblasts (MEFs) in S phase was significantly increased. Ectopic INCA1 expression reduced proliferation and colony formation of proliferating cells such as primary bone marrow cells, HeLa, HuTu80 and 32D cell lines. Serum starvation rapidly induced and mitogenic signals inhibited Inca1 expression providing a further link to cell cycle regulation. To identify the molecular mechanism of cell cycle regulation by Inca1, we investigated the influence of Inca1 on the direct inhibition of CDK2. In spleen lysates from Inca1-deficient mice, cellular CDK2 kinase activity towards Histone H1 was significantly induced compared to lysates of wildtype littermates. In in vitro kinase assays, recombinant INCA1 strongly inhibited CDK2 activity. In addition, we hypothesized that other cyclin kinase inhibitors (CKI) could partially compensate in vivo for the loss of Inca1 function. p21cip1/waf1 mRNA and protein expression were induced in Inca1−/ − MEFs compared to wildtype cells hinting at a partial compensation of the loss of Inca1 by induction of p21. Loss of Inca1 combined with p21 knockdown synergistically increased S-phase. These results indicate that Inca1 could be functionally related to p21 and that the rather mild phenotype observed in Inca1−/ − mice and the modest differences in Cdk activity observed in cell lysates lacking Inca1 could be due to compensatory induction of the CKI p21. In summary, loss of Inca1 increased cell proliferation, replating efficiency, S-phase progression, and Cdk2 activity whereas gain of Inca1 suppressed these cell functions. Inca1 expression was induced during cell cycle arrest. We conclude that Inca1 could be a novel cell cycle suppressor regulating the quiescence of HSCs through the inhibition of Cdk2.