ALBERT

Description: The immunoprivilege status characteristic of human amnion epithelial cells (hAECs) has been recently highlighted in the context of xenogenic transplantation. However, the mechanism(s) involved in such regulatory functions have been so far only partially been clarified. Here, we have analyzed the expression of HLA-Ib molecules in isolated hAEC obtained from full term placentae. Moreover, we asked whether these molecules are involved in the immunoregulatory functions of hAEC. Human amnion-derived cells expressed surface HLA-G and HLA-F at high levels, whereas the commonly expressed HLA-E molecule has been measured at a very low level or null on freshly isolated cells. HLA-Ib molecules can be expressed as membrane-bound and soluble forms, and in all hAEC batches analyzed we measured high levels of sHLA-G and sHLA-E when hAEC were maintained in culture, and such a release was time-dependent. Moreover, HLA-G was present in extracellular vesicles (EVs) released by hAEC. hAEC suppressed T cell proliferation in vitro at different hAEC:T cell ratios, as previously reported. Moreover, inhibition of T cell proliferation was partially reverted by pretreating hAEC with anti-HLA-G, anti-HLA-E and anti-β2 microglobulin, thus suggesting that HLA-G and -E molecules are involved in hAEC-mediated suppression of T cell proliferation. Finally, either large-size EV (lsEV) or small-size EV (ssEV) derived from hAEC significantly modulated T-cell proliferation. In conclusion, we have here characterized one of the mechanism(s) underlying immunomodulatory functions of hAEC, related to the expression and release of HLA-Ib molecules.

Description: Growth Factor Independence 1 (GFI1) is a hematopoietic transcription factor that plays a crucial role in the development of myeloid precursor cells. Its variant single nucleotide polymorphism called GFI136N (whereby Serine at position 36 is replaced by Asparagine) has been described to play a role in acute myeloid leukemia (AML) development. The prevalence of this variant is about 5-7% in the healthy Caucasian populations. Patients with myelodysplastic syndrome (MDS) show disturbed bone marrow function and are at risk to develop AML. Identifying prognostic markers and potential targets is essential for improved MDS therapy. We explored how GFI136N influences onset and progression of MDS and investigated the biological mechanisms behind our findings. To examine the role of GFI136N with regard to progression of MDS to AML, we characterized the status of GFI1 in 201 German-, 350 US- and 86 Dutch MDS patients. Our results revealed that 11-13% of patients were heterozygous for GFI136N compared to 5-7% in the healthy population. Patients carrying GFI136N also showed a 2-3-fold higher risk of AML-development and inferior leukemia-free survival than patients carrying the common variant GFI136S. The onset of MDS and AML tended to be at younger ages for GFI136N carriers and they did not respond to demethylating therapy to the same extent as GFI136S homozygous MDS patients. Whole exome sequencing revealed that patients carrying GFI136N had mutations in epigenetic modifiers such as EZH2 and expressed higher levels of different oncogenes such as HOXA9. To dissect the mechanisms behind these findings and to study the role of GFI136N in AML development, we used GFI136N and GFI136S knock-in mice that expressed either of the two human variants instead of murine Gfi1. We transduced hematopoietic progenitor cells from GFI136S or GFI136N expressing mice with retroviral vectors overexpressing oncofusion proteins AML1-ETO9a or MLL-AF9, which can be recurrently found in AML patient cohorts. GFI136N expressing cells that had been transduced with either AML1-ETO9a or MLL-AF9 generated significantly more cells and colonies than GFI136S controls. In order to confirm our findings in vivo, we used three different AML and MDS mouse models crossed to mice carrying either GFI136N or the common GFI136S. As a first in vivoapproach, we used a mouse strain that expresses the human oncofusion protein CBFB-MYH11 (inv(16)) frequently found in AML patients. Mice carrying GFI136N and expressing CBFB-MYH11 showed a significantly accelerated onset of AML compared to mice carrying GFI136S (p=0.004). We found similar results in a second AML mouse model expressing MLL-AF9. Using the NUP98-HOXD13 MDS mouse model a marginally significant decrease in leukemia-free survival was observed in mice carrying GFI136N. To investigate molecular mechanisms that could cause the observed effects of GFI136N, we used ChipSeq and RNASeq. Our results revealed that GFI136N lineage negative cells show a genome-wide higher degree of H3K4 methylation and H3K9 acetylation than cells carrying GFI136S. GFI1 recruits among others LSD1 and HDAC to demethylate H3K4 and deacetylate H3K9 and thus GFI136N fails to initiate epigenetic changes previously shown for GFI1, leading to higher expression of various oncogenes such as HoxA9 and Kras. A number of MDS and AML patients are treated with HDAC and LSD1 inhibitors, but based on the above data we would predict that this therapy would likely fail in GFI136N patients and that therapy with histone acetylase (HAT) inhibitors or histone methyltransferase inhibitors would be more beneficial for these patients. To test this hypothesis in vitro, we treated the above-described GFI136N and GFI136S expressing AML1-ETO9a or MLL-AF9 leukemic cells with HAT or HDAC inhibitors. HAT inhibitors inhibited growth of preleukemic GFI136N expressing cells more efficiently than HDAC inhibitors. Similarly in vivo, we transplanted irradiated mice with GFI136N or GFI136S expressing MLL-AF9 leukemic cells. We found that HAT inhibitors tended to prolong the survival of mice transplanted with GFI136N leukemic cells more than mice transplanted with GFI136S leukemic cells. In summary, GFI136N is a novel prognostic marker for MDS patients that predisposes to AML likely via epigenetic changes at different oncogenes. We also suggest that HAT inhibitors could be more beneficial to GFI136N carrier patients thus enabling a more individualized therapy. Disclosures Platzbecker: TEVA: Research Funding, Speakers Bureau.

Description: The proper differentiation of hematopoietic stem cells is regulated by a concert of different so called transcription factors. Growth Factor Independence 1b (Gfi1b) is a repressing transcription factor, which is pivotal for the proper emergence and maturation of erythrocytes and platelets. Furthermore, Gfi1b controls quiescence as well as cell cycle progression of hematopoietic stem cells and early progenitor cells. It has been shown for other transcription factors that a disturbed function of these transcription factors can be the basis of malignant diseases such acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). MDS is characterized by disturbed differentiation of one or several hematopoietic lineages. The accumulation of malignant blast cells, which are arrested in their development, is a key feature of AML. Since transcription factors play a role in AML development, we sought to investigate whether Gfi1b might also play a role in the development and progression of AML. Based on published gene expression arrays and own patient samples, we observed that Gfi1b is expressed at a lower level in leukemic blasts and leukemic stem cells compared to the non-malignant control cells. We correlated Gfi1b expression level in blast cells of patients from Essen and we found out that patients with high Gfi1b levels had a poor prognosis and an increased risk of relapse. In contrast low levels of Gfi1b expression were associated with a good prognosis. To test how different levels of Gfi1b might influence initiation of AML we have mouse strains available expressing Gfi1b at different expression levels. We have Gfi1b wt mice with one allele of Gfi1b deleted (Gfi1b het) and Gfi1b conditional mice, in which the expression of Gf1b (Mx Cre tg Gfi1b fl/fl) can be abrogated after injecting these mice with pIpC. To explore the role of Gfi1b in leukemia development, we used different murine AML models, resembling human leukemia. First these mice were crossed with Nup98HoxD13 transgenic mice, a mouse strain that develops a disease similar to the human MDS. We observed that Gfi1b heterozygosity (n=15) accelerated AML development (p=0,03) compared to wt mice (n=16). More importantly, complete absence of Gfi1b (n=8) results in a substantially earlier onset of AML, with a median survival time of about 50 days (p=0.002). To confirm our findings, we used a different murine AML model. Recurrent so called oncofusion proteins such as AML1-Eto9a, CBFbeta-Myh11 or MLL-AF9 are characteristic for certain subtypes of AML. We transduced Lineage negative (Lin-) bone marrow cells from wt, Gfi1b heterozygous (Gfi1b het) and Gfi1b deficient (MxCre Gfi1b fl/fl) mice with retroviruses encoding either AML1-Eto9a or MLL-AF9 oncofusion -proteins. Transduced Gfi1b heterozygous or Gfi1b deficient cells generated more colonies and higher cell number than wt transduced cells. We also used mice transgenically expressing CBFbeta-Myh11. Deletion of Gfi1b accelerated leukemia formation in these mice compared to mice in which Gfi1b was still expressed. On a molecular level, we found that loss of Gfi1b leads to increased levels of ROS level. It has been shown by other groups, that increased levels of Gfi1b contribute to leukemia development. In addition, Gfi1b represses the expression of Integrin beta 3 (ITGB3). Absence of Gfi1b leads to higher expression level of ITGB3. ITGB3 has been shown to promote growth and expansion of leukemic stem cells, which play an important role in AML development. Thus we report here that Gfi1b acts as a novel tumor suppressor in AML development. Disclosures: No relevant conflicts of interest to declare.

Description: The receptor tyrosine kinase Axl belongs to the TAM (Tyro-3, Axl and Mer) family and is involved in the progression of several human malignancies including chronic lymphocytic leukemia (CLL), where it is has been found to be overexpressed in comparison to normal B-cells. An increasing body of evidence suggests that Axl acts as an oncogene which increases the survival, proliferation, metastatic potential and chemotherapy resistance of tumor cells. Hence, it has been recently identified as a potential therapeutic target in a wide range of tumor entities with deregulated Axl expression including prostate cancer, glioma, lung cancer and CLL. Here, we investigated two different Axl inhibitors for their ability to inhibit the migratory capacity and survival of leukemic cells in preclinical CLL models. In vitro studies: We measured soluble Axl plasma concentrations by enzyme-linked immunosorbent assay (ELISA) in 71 CLL patients and 24 healthy donors. Soluble Axl levels were not significantly higher in CLL patients compared to healthy donors (p=0. 11). However, in CLL patients high sAxl plasma concentrations were differentially expressed with some patients exhibiting normal and others elevated plasma concentrations. The latter showed an association with shorter time to first treatment (p=0.0005) and several poor prognostic markers (e.g. CD38, FISH cytogentics, Binet stage). Freshly isolated PBMC (〉90% CD5+CD19+) from CLL patients were incubated in serum free medium for 48h containing concentrations series of 2 different Axl inhibitors: BMS777607, a previously published inhibitor of the MET kinase family, and LDC2636, a novel inhibitor of the TAM receptor tyrosine kinase (RTK) family with high affinity to Axl. Viability of CLL cells was assessed by trypan blue staining and flow cytometry employing annexin V staining. Cellular polarization was analyzed by time-lapse microscopy. We detected cytotoxic effects in a patient-dependent manner that were more prevalent in LDC2636 as compared to BMS777607 treated cells (IC50= 0.21 µM vs. 2.88 µM, p

Description: The differentiation of hematopoietic stem cells to mature cells is essential for the function of the hematopoietic system. Disturbance of this process can lead to the emergence of Acute Myeloid Leukemia (AML). AML is characterized by an accumulation of immature, malignant blasts, which disturb the function of the “normal” hematopoietic cells. The differentiation to myeloid cells is regulated among others by transcription factors. Growth factor independence 1 (GFI1) is such a hematopoietic transcription factor regulating the differentiation of myeloid cells. We sought to investigate whether different Gfi1 levels are causative for emergence of AML and how different levels of Gfi1 might influence the prognosis of patients. Using published expression array data, we observed that Gfi1 is expressed at a lower expression level in blast cells and in leukemic stem cells compared to the control non-malignant cells and stem cells. We then correlated Gfi1 expression level in blast cells of patients from different centers in with the event free survival. In Essen and Dresden (Germany), low expression levels in blast cells were (n=39) associated with an inferior prognosis (EFS 9 months for low expression compared to 42 months; p=0.0095). We confirmed our observation with an independent cohort from Rotterdam and Nijmegen. Patients with low Gfi1 expression (n=32) had an inferior event free survival (9 months) compared to patients with higher Gfi1 levels (n=144; 17 months; p=0.02). To further investigate how different levels of Gfi1 might influence initiation and progression of leukemia, we used mice expressing Gfi1 at different levels, i.e. Gfi1 deficient mice (Gfi1KO), mice heterozygous for Gfi1 (Gfi1 het) or mice expressing Gfi1 only at 20% of normal Gfi1 expression levels (Gfi1KD). We used different murine AML models to examine the role of Gfi1 in AML development. First we crossed these mice with Nup98HoxD13 mice that recapitulate MDS disease course. We observed that knockdown of Gfi1 (Gfi1KD n=15, P=0.05) and heterozygosity of Gfi1 (Gfi1 het) (n=12) accelerated AML development and were associated with higher blast cell number compared to Gfi1 wt mice (n=16). Interestingly, complete loss of Gfi1 (Gfi1KO, n=16) inhibited leukemia development. To confirm our findings, we used an independent approach. It has been shown previously that enforced retroviral expression of certain onco-fusion proteins such as MLL-AF9 or AML1-ETO9a or proteins such as MN1 can cause AML in mice. To this end we transduced lineage negative (Lin-) cells from the different mouse strains with retroviruses overexpressing these different proteins and plated the Lin-cells in methycellulose media. Similar to above, transduced Gfi1 KD cells generated more colonies and proliferated at higher levels than wt or Gfi1 KO cells (ranging between 1,5 to 4 fold KD compared to wt, depending on oncofusionprotein or oncogene, p=0.05). We also transplanted these cells into lethally irradiated mice. Again, mice transplanted with MLL-AF9 transduced Gfi1 KD cells (n=6) developed leukemia faster than mice transplanted with transduced wt (n=8; p= 0.05). We hypothesized that if lower expression of Gfi1 promotes leukemia formation, then overexpression of Gfi1 should inhibit leukemia formation. To this end we transduced Lin neg cells simultaneously with retroviruses overexpressing either MLL-AF9 or AMLETO9a and either Gfi1 or an empty vector. The cells were then plated in methycellulose and cells overexpressing Gfi1 generated fewer colonies (between 3-5 fold less, depending on oncogene, p=0.01) than cells transduced with an empty control vector. Thus, up to now our data suggests that decreased levels of Gfi1 influence prognosis of AML development and are involved in the pathogenesis of AML. On a molecular level, we found that knock-down and complete loss of Gfi1 leads to deregulation of genes in AML development such as HoxA9. However, whereas Gfi1KD cells show a normal response to apoptotic events, complete loss of Gfi1 is associated with a very high level of spontaneous apoptosis, possibly explaining why knock-down but not complete loss of Gfi1 accelerates AML development. In summary we demonstrate that Gfi1 plays a crucial role in AML development depending on the expression level, which in turn might explain the role of Gfi1 in human AML. Disclosures: No relevant conflicts of interest to declare.

Description: Myelodysplastic syndromes (MDS) are characterized by disturbances in the development of different blood lineages, which can progress to AML. The knowledge about the factors predisposing the development of AML is sparse. Growth factor independence 1 is a transcription factor regulating the differentiation of myeloid cells. Previously a Single Nucleotid Polymorphism (SNP) of Gfi1, denominated Gfi136N, had been described. This variant is characterized by a SNP leading to the replacement of Serine at position 36 to an Asparagine. This SNP is found in 5-7% of all Caucasians and in 10-15% of all AML patients. We sought to investigate whether Gfi136N could be a novel predictive marker for the development of AML in MDS patients. To this end, we characterized 201 patients with regard to presence of the Gfi136N variant. Patients were recruited from different German centers treating MDS patients, i.e. Essen, Düsseldorf and Dresden. Within this cohort, about 10% were heterozygous for Gfi136N. There was no difference between MDS patients heterozygous for Gfi136N or homozygous for Gfi136S with regard to age, sex, cytogenetic or IPSS score. Presence of Gfi136N significantly increased incidence (Odds ratio 2 fold) and shortened latency to AML progression (around 2 years for Gfi136N heterozygous patients compared to around 6 years for Gfi136S homozygous patients (p=0.001). To further investigate the role of Gfi136N in AML development, we generated mice expressing either the wildtype form of human Gfi1 (Gfi136S) or Gfi136N. We mated these mice with mice expressing the Nup98HoxD13 transgene. Mice expressing Nup98HoxD13 develop a MDS like disease and about 20-30% progress to AML. Mice with Nup98HoxD13 and Gfi136N alleles (n=10) developed AML with a higher incidence (60% compared to 20%) and shorter latency (200 days compared to 340 days) than mice with Nup98HoxD13 and Gfi136S alleles. (n=8, p=0.05) To confirm our finding, we used additional murine AML models resembling human AML cells. MLL-AF9 and AML1-ETO9a are recurrent so called oncofusionproteins, which are charateristic for different human AML subtypes. By retroviral transduction of murine Lineage negative (Lin neg) cells, murine leukemia resembling human AML can be generated. We transduced Lin neg cells from Gfi136S and Gfi136N mice with retroviruses expressing the onocfusionporteins MLL-AF9 or AML1-ETO9a. After seeding in semi-solid medium, transduced Gfi136N cells generated more colonies with a higher cell number than transduced Gfi136S cells (2-4 fold more cells or colonies, depending on the oncofusionprotein, p=0.05). In summary, our data suggest that Gfi136N is a novel predictive marker for AML development among MDS patients which can be recapitulated in mice. To investigate the reason behind this observation, we analyzed lineage negative ckit pos blood cells from Gfi136N or Gfi136S homozygous mice. Genome-wide analysis of histone modification showed that mice expressing the 36N variant display globally higher levels of diMeH3K4 and AcH3K9 activation marks with a significant positive correlation between them. We analyzed the genes, which had a higher level of activation marks in Gfi136N cells compared to Gfi136S. We found that pathways involved in cytokine signaling, hematopoietic lineage development and AML genesis were overrepresented among these genes. Thus we show that Gfi136N might play a crucial role in AML development of MDS patients by inducing epigenetic changes, which promote AML development. Disclosures: Germing: Celgene: Honoraria, Research Funding; Jansen-Cilag: Honoraria; Novartis: Research Funding; GSK: Research Funding; Amgen: Research Funding. Platzbecker:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.