ALBERT

Description: Processing of pre-miRNA through Dicer1 generates an miRNA duplex that consists of an miRNA and miRNA* strand. Despite the general view that miRNA*s have no functional role, we further investigated miRNA* species in 10 deep-sequencing libraries from mouse and human tissue. Comparisons of miRNA/miRNA* ratios across the miRNA sequence libraries revealed that 50% of the investigated miRNA duplexes exhibited a highly dominant strand. Conversely, 10% of miRNA duplexes showed a comparable expression of both strands, whereas the remaining 40% exhibited variable ratios across the examined libraries, as exemplified by miR-223/miR-223* in murine and human cell lines. Functional analyses revealed a regulatory role for miR-223* in myeloid progenitor cells, which implies an active role for both arms of the miR-223 duplex. This was further underscored by the demonstration that miR-223 and miR-223* targeted the insulin-like growth factor 1 receptor/phosphatidylinositol 3-kinase axis and that high miR-223* levels were associated with increased overall survival in patients with acute myeloid leukemia. Thus, we found a supporting role for miR-223* in differentiating myeloid cells in normal and leukemic cell states. The fact that the miR-223 duplex acts through both arms extends the complexity of miRNA-directed gene regulation of this myeloid key miRNA.

Description: Abstract 501 The functional roles of microRNAs in the development of acute myeloid leukemia (AML) are not yet clear. Due to its myeloid-specific expression, miR-223 has been one of the most-investigated miRNAs in normal and malignant hematopoiesis. However, the role of miR-223 in myeloid differentiation is not completely understood, as contradicting data exists. Genetic depletion of miR-223 led to a significant increase of myeloid progenitor cells as well as circulating hyperreactive neutrophils. Here, we investigate the role of miR-223 in the development of AML in vivo, using retroviral overexpression models of Hoxa9 with Meis1 or MN1 as two potent models of leukemic transformation in a miR-223+/+ or miR-223−/− background. In contrast to the observed high level expression of miR-223 in human CD34- bulk AML cells (p=0.0106), we could show that miR-223 was dispensable for the development of AML and did not impact on either the leukemic stem cell frequency nor the AML cell phenotype in Hoxa9-Meis1 AML cells. While these findings reveal that miR-223 is not necessary for leukemic transformation in highly aggressive AML models, we became interested if miR-223 functions rather as modulator of disease progression, especially at the early development of AML. Therefore, we investigated the role of miR-223 with regards to differentiation and self-renewal in two preleukemic model systems by retrovirally infecting miR-223−/− and miR-223+/+ BM cells with AML1-ETO and Hoxa9 respectively. Characterization of these models demonstrated that miR-223 expression is a determinant of differentiation, as miR-223−/− preleukemic cells exhibit a significant lower Mac-1 expression (p=0.0003). However, in contrast to normal miR-223−/− BM cells, which show a significantly higher colony forming capacity in methylcellulose compared to miR-233+/+ BM cells, the colony forming capacity of miR-223−/− or miR-223+/+ preleukemic cells did not significantly change. These findings demonstrate that miRNA miR-223 is hierarchically expressed in AML cells, and functionally link miR-223 to impaired differentiation rather than increased self-renewal in the initiation of AML. This indicates that miR-223 is more likely a fine tuner of leukemic development than a potent tumor suppressor or oncogenes as suggested in the literature. However, it still remains to be shown if the presence of miR-223 influences the susceptibility of preleukemic cells to convert into leukemia initiating cells. Disclosures: No relevant conflicts of interest to declare.

Description: Synergistic deregulation of HOXA9 and the HOX-gene cofactor MEIS1 is a commonly observed phenomenon in acute myeloid leukemia (AML). The leukemogenic potential of aberrant Hoxa9 and Meis1 expression has been shown in several AML models. However, the molecular mechanisms behind Hoxa9- and Meis1-induced leukemogenesis are still not well understood. In order to identify functionally relevant Meis1-induced microRNAs (miRNA), we profiled the global miRNA expression using a Hoxa9-Meis1 murine AML progression model. This two-step model allowed us to quantify miRNAs at a pre-leukemic stage through the overexpression of the proto-oncogene Hoxa9 (Hoxa9/ctrl), as well as after full leukemic transformation through co-overexpression of Hoxa9 and Meis1 (Hoxa9/Meis1). The pre-leukemic stage is characterized by in vitro immortalization without in vivo engraftment, whereas the transplanted leukemic cells induce full-blown AML in vivo. MiR-155 turned out to be one of the most significant differentially expressed miRNA species and its upregulation was independently validated in Hoxa9/Meis1 cells by qRT-PCR. Subsequent analysis of various AML subtypes (CN-AML, t(11q23), t(8;21), t(15;17), n=38) showed significantly elevated levels of miR-155 in CN-AML with NPM1mut (n=10, p

Description: Abstract 2874 Although novel treatment options have improved response rates and survival in CLL, the portion of cases with refractory disease remains considerably high and management is challenging. The humanized anti-CD52 monoclonal antibody alemtuzumab is standard treatment in refractory CLL and shows efficacy irrespective of the underlying genomic aberrations or mutation status of TP53. However, only about half of all refractory cases can be explained by TP53 mutation/deletion and treatment remains unsatisfactory. To elucidate pathogenic and prognostic factors, we performed microRNA expression analyses on a set (n=42) of fludarabine-refractory CLL patients treated within the multicenter phase II CLL2H trial of the GCLLSG (3×30 mg alemtuzumab s.c. per week, max 12 weeks). Profiling was done with the Agilent microRNA microarray platform based on the Sanger miRBase Release 14.0. Statistical analysis was performed with the R software version 2.15.1. Normalization of the arrays was conducted by using the least-variant set (LVS) of genes method and by quantile normalization. When filtering the expression data based on standard deviation (sd〉0.5), we found 131 microRNAs showing greatest variability in expression levels across all arrays. Subsequent unsupervised bootstrap clustering (mean centered; distance: pearson correlation; agglomeration rule: average linkage) resulted in two stable major clusters. Cluster 1 contained more cases with p53 defect which were observed in 56% (TP53 mutation 7/16, 17p deletion 6/16) vs. 27% (TP53 mutation 6/26, 17p deletion 5/26). Cluster 2 was slightly enriched for IGHV unmutated cases in 61% (16/26) vs. 50% (8/16). Median levels of thymidine kinase were 31.6 U/L in cluster 1 and 39.6 U/L in cluster 2, beta2-microglobulin levels were similar in both clusters with a median of 4.3 and 4.4 mg/L. Expression of miR-34a, a microRNA known to execute p53-dependent cell cycle arrest, as well as induction of senescence and apoptosis, was predominantly low in cluster 1. Cases with low miR-34a expression in cluster 1 and without altered p53 (no 17p deletion and/or TP53 mutation) showed a selective upregulation of microRNAs known to be involved in proliferation, neoangiogenesis and inhibition of apoptosis by targeting genes like CDKN1A, E2F, BIM and PTEN. Interestingly, these microRNAs have been reported to be repressed by p53 under physiologic conditions, pointing to alternative mechanisms of p53 pathway inactivation. With regard to treatment response, cluster 1 was significantly enriched for better response (partial response (PR) n=9 (56.25%); stable disease (SD) n=5 (31.25%); progressive disease (PD) n=2 (12.5%)). In contrast cluster 2 (n=26) was dominated by cases with inferior response (PR n=4 (15%); SD n=13 (50%); PD n=9 (35%)). Progression free survival (PFS) was significantly longer in cluster 1 (median 331 days vs. 145 days, p=0.0171, Figure 1). Overall survival was in trend longer in cluster 1 (median 760 days vs. 513 days, p=0.2480). In conclusion, microRNA expression in fludarabine-refractory CLL shows high variability likely induced by independent mechanisms of resistance. Altered microRNA expression might mimic effects similar to TP53 mutation or 17p deletion even in situations when p53 itself is not functionally disrupted. Of note, unsupervised clustering identified two major clusters that showed a surprising prognostic relevance in fludarabine-refractory CLL treated with alemtuzumab. Figure 1: Progression free survival of refractory patients treated with alemtuzumab based on major clusters after unsupervised clustering. Figure 1:. Progression free survival of refractory patients treated with alemtuzumab based on major clusters after unsupervised clustering. Disclosures: Stilgenbauer: Genzyme: Honoraria, Research Funding.

Description: MicroRNAs (miRNAs) are essential for maintenance and differentiation of normal hematopoietic cells and their dysregulation is strongly implicated in leukemias. In order to identify tumor suppressor miRNAs in the context of hematological malignancies, we performed two complementary miRNA expression screenings in normal hematopoiesis as well as in childhood and adult acute myeloid leukemias (AML). We reasoned that tumor suppressor miRNAs are upregulated in mature myeloid cells, as compared to normal hematopoietic stem and progenitor cells (HSPCs), and downregulated in AML. Based on this screening strategy, we identified the miR-193 family members as potent suppressors of HSPC activity and AML growth. During normal hematopoiesis mmu-miR-193a-3p is exclusively expressed in mature myeloid cells and absent in normal HSPCs. Accordingly, in a cohort of 165 pediatric AML patients hsa-miR-193b-3p was broadly repressed throughout the cytogenetically characterized subgroups. In addition, in a cohort of 43 adult AML patients, its homolog hsa-miR-193a-3p was significantly upregulated in APL cases (p=0.0025, n=7) compared to bone marrow from healthy donors (n=5). To assess the impact of the miR-193 family members on AML maintenance and development, we lentivirally expressed miR-193a/b in the MLL-rearranged cell lines ML2 and THP1, which induced monocytic differentiation in concert with calcitriol treatment, measured by CD11b/CD14 expression (p=0.024). Consistently, enforced miR-193-expression led to a significant growth disadvantage in ML2 and THP1 cells (p=

Description: The miR-106a-363 cluster, encoding six miRNAs (miR-106a, miR-18b, miR-20b, miR-19b, miR-92a and miR-363), is a paralogue of the oncogenic miR-17-92a polycistron and its role in leukemia is at present largely unknown. We aimed to investigate the putative oncogenic role of the miR-106a-363 cluster in adult acute myeloid leukemia (AML) and to dissect the contributions of its individual members to disease formation and progression. First, we analyzed the expression of each miRNA in AML patient samples as well as their clinical relevance. To determine the association of the miR-106a-363 cluster in AML with active disease, we quantified all six miRNAs individually in AML patient samples at initial diagnosis (n=33) and in AML patients in complete remission after chemotherapy (n=6). Hereof, miR-106a-5p, miR-19b-3p and miR-92a-3p levels were significantly lower in remission samples (p=0.0015, p=0.0013 and p=0.0004, respectively), confirming that these miRNAs are upregulated in AML. Stratifying AML patients within the LAML miRNA-Seq dataset of The Cancer Genome Atlas (TCGA) Research Network (n=187) (Ley et al., NEJM, 2013) according to their cytogenetic risk group demonstrated that all members of the cluster, except for miR-18b-5p, significantly associated with adverse cytogenetics. In addition, with the exception of miR-18b-5p, all members associated with an inferior overall survival (OS) in AML patients within the TCGA-LAML dataset, further supporting a pro-leukemogenic role for the cluster. Of note, miR-106a-5p was the most abundantly expressed unique miRNA of the polycistron, both in the TCGA patient cohort and in 11 myeloid leukemia cell lines quantified by quantitative real-time PCR (qRT-PCR). Since the miR-106a-363 cluster is associated with high risk AML, we hypothesized that increased levels of the entire cluster as well as individual members would significantly shorten the survival time in a murine transplantation model mimicking aggressive AML. Therefore, we engineered transplantable, primary murine AML cell lines based on retroviral overexpression of Hoxa9 and Meis1 exhibiting a median disease latency of 39 days (n=14) after syngeneic transplantation in mice. Enforced lentiviral expression of miR-106a-363 (n=13, p

Description: Acute Myeloid Leukemia (AML) is driven by cell populations with stem cell-like characteristics, so called leukemia stem cells (LSC). The transcription factor Meis1 is one of the critical regulators of LSC and is capable to rapidly induce AML in murine models in the context of Hox gene overexpression. Despite sophisticated studies identifying Hox- and Meis1-regulated genes, the knowledge about their impact on intracellular signaling pathways and its functional consequences is still limited. Since Hox and Meis1 gene overexpression is often found in high risk AML and since both factors are currently considered as undruggable, we aimed to elucidate their role in regulating intracellular signaling and to investigate, if cells transformed by Hoxa9 and Meis1 are addicted to certain signaling processes. To characterize the effect of Meis1 in the context of Hox gene overexpression on protein expression and intracellular signaling, we have performed a comprehensive (phospho)proteomic analysis and correlated it with transcriptome sequencing data. Our analysis revealed that Meis1 upregulates expression of spleen tyrosine kinase (Syk) without affecting its mRNA expression level. This was confirmed in patient-derived AML cells. By global analysis of microRNA expression and subsequent functional analyses, we could identify the downregulation of miR-146a, which turned out to be PU.1-dependent, as a mediator for this post-transcriptional upregulation of SYK in Hoxa9/Meis1 overexpressing cells. To further investigate, if an activation of Syk signaling can mimic Meis1 in inducing leukemia in our murine AML transplantation model, we overexpressed Syk in Hoxa9-transformed myeloid progenitors and found that this resulted in an acceleration in leukemia development comparable to the acceleration observed upon combined Hoxa9/Meis1 overexpression. We also found that Syk overexpression resulted in an increased expression of Meis1 and an induction of Meis1-dependent gene expression signatures. Notably, Hoxa9/Meis1-transformed cells also exhibited a remarkable sensitivity to Syk inhibition and SYK knockdown in vitro and in vivo, while Hoxa9-transformed cells did not. In summary, we identified a previously unknown signaling loop between Meis1 overexpression and Syk signaling involving miR-146a as a regulator of SYK expression. Hence, we believe that Syk inhibition by small molecules might be a potential therapeutic option for AMLs particularly in the context of Hox/Meis1-overexpression. Disclosures Berg: Astellas: Other: Travel Funding; Alexion: Other: Travel Funding; Celgene: Other: Travel Funding.

Description: Background: The nucleophosmin 1 (NPM1) gene is not only commonly mutated in acute myeloid leukemia (AML), but also encodes several linear splice isoforms, one of which was recently shown to be of prognostic importance. Furthermore, circular RNAs (circRNAs) are transcribed from the NPM1 gene which demands further investigation with regard to function in normal hematopoiesis and impact on leukemogenesis. Aims: We aimed to investigate circRNAs derived from NPM1 and gain insights into their regulation and function. Additionally, we wanted to determine changes in the circular RNAome in the course of hematopoietic differentiation and leukemic transformation. Methods: Circular NPM1 transcripts were detected by PCR and sequenced in leukemic cell lines (n=7) and healthy control samples (n=3, peripheral blood-derived mononuclear cells). Expression of hsa_circ_0075001 and total NPM1 was measured in a cohort of 23 NPM1 wildtype (NPM1wt) and 23 NPM1 mutated (NPM1mut) AML patients via quantitative real-time PCR (qPCR), and Affymetrix U133plus2 microarray data was set in relation to the expression levels. Principal component analysis (PCA) was conducted to identify groups with similarities in gene expression patterns and differentially expressed genes were subjected to pathway analysis. Next, ribosomal RNA-depleted RNA-seq was performed for 5 NPM1mut and 5 NPM1wt AML cases, as well as 10 healthy control samples derived from 4 FACS-sorted myeloid differentiation stages (myeloblasts, promyelocytes, metamyelocytes and neutrophils). PCA and unsupervised hierarchical clustering were performed based on circRNA expression. Results: We detected and sequenced multiple circular NPM1 transcripts (n=23) in leukemic as well as in healthy control cells. As hsa_circ_0075001 showed differential expression between different AML cell lines in a semi-quantitative PCR analysis, quantification in 46 AML patients via qPCR was performed. This analysis revealed that total NPM1 and hsa_circ_0075001 expression were independent of the NPM1 mutational status. Furthermore, the hsa_circ_0075001 expression status defined distinct leukemia subgroups characterized by similarities in gene expression as determined by PCA. For example, differentially expressed genes between high versus low hsa_circ_0075001 expression groups (dichotomized at the median) were significantly enriched in components of the Toll-like receptor (TLR) signaling pathway, which was downregulated in patients with high hsa_circ_0075001 expression. Expression of hsa_circ_0075001 correlated positively with total NPM1 expression, and RNA-seq analysis further revealed a global correlation of circRNA and parental gene expression. In total, in our cohort circRNAs were found for 19 % of all expressed genes. PCA based on circRNA expression illustrated that immature and mature hematopoietic cells, as well as NPM1wt and NPM1mut AML samples, exhibit distinct circRNA signatures (Figure 1). Thus, circRNA expression seems to play a role during differentiation of normal hematopoietic cells, but also seems to be severely deregulated in AML. Figure 1: Altered circular RNA expression in AML patients compared to healthy control samples. Principal component analysis (PCA) of circRNA expression data of 5 NPM1mut patients (red), 5 NPM1wt patients (green), and 10 healthy control samples, of which 4 were derived from immature (blue) and 6 from more mature myeloid differentiation stages (purple). Data was generated via RNA-Seq and reads derived from circRNAs were aligned and quantified using STAR, and normalized and transformed using DESeq2. PCA was performed based on 500 genes with the highest variance of circRNA expression across all samples. Conclusions: circRNAs transcribed from the NPM1 gene showed differential expression in AML cell lines and healthy cells, and higher hsa_circ_0075001 expression defined an AML subgroup characterized by downregulation of the TLR signaling pathway. These findings provide evidence for the relevance of circular NPM1 transcripts and add another level of complexity to the multifaceted gene NPM1. In general, circRNA expression seems to be involved in the regulation of hematopoietic differentiation, which is in line with previous observations, but, based on distinct circRNA expression profiles in AML, they might also play a significant pathogenic role in leukemic transformation. Figure 1 Figure 1. Disclosures Paschka: Celgene: Honoraria; Pfizer Pharma GmbH: Honoraria; Bristol-Myers Squibb: Honoraria; Medupdate GmbH: Honoraria; Novartis: Consultancy; ASTEX Pharmaceuticals: Consultancy.

Description: Background: Genetic rearrangements that fuse the mixed lineage leukemia (MLL) gene, now termed KMT2A, to one of a variety of partners are seen in 5% - 20% of acute myeloid leukemia (AML). MLL -fusions are especially common in childhood AML and many of them are associated with poor prognosis. The MLL -fusions perturb transcription through different mechanisms and they are often associated with high expression of the transcription factors HOXA9 and MEIS1. Based on a micro-RNA screen in an AML mouse model mimicking the step-wise development of aggressive AML we have found that concurrent Hoxa9 and Meis1 overexpression is associated with upregulation of micro-RNA-155 (miR-155). Expression of miR-155 was also found to be higher in bone marrow samples from patients with MLL- AML compared with bone marrow from healthy donors (p