The contribution of molecular alterations in bone marrow mesenchymal stromal cells (BM-MSC) to the pathogenesis of acute myeloid leukemia (AML) is poorly understood. Thus we assessed genome-wide genetic, transcriptional and epigenetic alterations in BM-MSC derived from AML patients (AML BM-MSC). Whole-exome sequencing (WES) of AML BM-MSC samples from 21 patients revealed a non-specific pattern of genetic alterations in the stromal compartment. The only mutation present in AML BM-MSC at serial time points of diagnosis, complete remission and relapse was a mutation in the PLEC gene encoding for cytoskeleton key player Plectin in one AML patient. Healthy donor controls did not carry genetic alterations as determined by WES. Transcriptional profiling using RNA sequencing revealed deregulation of proteoglycans and adhesion molecules as well as cytokines in AML BM-MSC. Moreover, KEGG pathway enrichment analysis unravelled deregulated metabolic pathways and endocytosis in both transcriptional and DNA methylation signatures in AML BM-MSC. Taken together, we report molecular alterations in AML BM-MSC suggesting global changes in the AML BM microenvironment. Extended investigations of these altered niche components may contribute to the design of niche-directed therapies in AML.

No abstract

No abstract

Overexpression of the oncogene ERG (ETS-related gene) is an adverse prognostic factor in acute myeloid and T-cell lymphoblastic leukemia (AML and T-ALL). We hypothesize that ERG overexpression is associated with primary drug resistance thereby influencing the outcome in leukemia. We previously reported a cell-line based model of ERG overexpression which induced a potentially chemo-resistant spindle shape cell type. Herein, we report a specific transcriptional gene signature for the observed spindle shaped morphology. Genes significantly over-expressed after ERG induction strongly resembled adhesive mesenchymal-like genes that included integrins (ITGA10, ITGB5, ITGB3, ITGA2B), CD44, and CD24. Interestingly, the mesenchymal-like signature was accompanied by the repression of DNA chromatin remodeling and DNA repair genes, such as CHEK1, EZH2, SUZ12, and DNMT3a. The ERG-induced mesenchymal-like signature positively correlated with TMPRSS2-ERG prostate tissues and invasive breast cancer mRNA expression datasets reflecting a general ERG-driven pattern of malignancy. Furthermore, inhibitors modulating ERG druggable pathways WNT, PKC, and AKT, and chemotherapeutic agent cytarabine revealed ERG-induced drug resistance. In particular, PKC412 treatment enhanced proliferative rates and promoted spindle shape formation in ERG-induced cells. Nilotinib and dasatinib were effective at abolishing ERG-induced cells. Moreover, ERG overexpression also led to an increase in double strand breaks. This report provides mechanistic clues into ERG-driven drug resistance in the poor prognostic group of high ERG expressers, provides insight to improved drug targeted therapies, and provides novel markers for a mesenchymal-like state in acute leukemia.

No abstract

1607 Poster Board I-633 INTRODUCTION Overexpression of the gene ERG (v-ets erythroblastosis virus E26 oncogene homolog) is an adverse prognostic factor in adult patients with acute T-lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). However, the underlying biology remains unknown. The aim of this study was to investigate the regulation of ERG expression by microRNAs (miRNAs) and to explore their potential role in acute leukemia and normal hematopoiesis. METHODS: A bioinformatic database search was carried out using the Targetscan, Pictar, and Human microRNA target tools to predict ERG regulating miRNAs. Verification of ERG as potential target of predicted miRNAs was performed by AMAXA transfection of miRNA precursor molecules in the myeloid leukemic cell line KG1a. After 24 hours (hrs) and 48 hrs total RNA was extracted using the Trizol reagent. Overexpression of the miRNAs was confirmed by TaqMan MicroRNA assays and ERG expression was determined by real-time RT-PCR. Moreover, specific binding of miRNAs to the 3'UTR of ERG was verified by luciferase reporter assays co-transfecting the ERG 3'UTR cloned into the psiCHECK-2 luciferase vector with miRNA precursor molecules. To investigate the expression of miRNAs during hematopoietic maturation, CD34 positive bone marrow cells from healthy individuals were in vitro cultured using the cytokines SCF and IL-3 (maintenance culture) with the addition of EPO or G-/GM-CSF. Cells were harvested after 3, 6, 9, 13, 16, and 20 days and miRNA expression levels were measured. The expression of miR-196a-1 and miR-196b was also studied in acute leukemias including bone marrow samples of adult patients with newly diagnosed T-ALL (n=105) and AML (n=34). RESULTS: By the database search, a total of 13 miRNAs were predicted to potentially regulate ERG and were further studied. Of these, only the miRNAs miR-196a-1 and miR-196b induced a significant reduction of ERG expression levels. After 24 hrs ERG was significantly down-regulated by 36% (after miR-196a-1 transfection) and by 42% (after miR-196b transfection) as well as after 48 hrs by 43% (after miR-196a-1 transfection) and by 47% (after miR-196b transfection) compared to the controls. The luciferase assays revealed a 30% and 40% luciferase activity reduction in miR-196a-1 and miR-196b transfected cells, respectively, compared to the miRNA-missense transfected cells. This confirmed the direct binding of these miRNAs to the ERG 3'UTR. During hematopoietic differentiation of normal CD34 positive progenitors, expression of miR-196a-1 was constant over time using the different cytokine conditions. In contrast, the expression of miR-196b decreased substantially during the in vitro differentiation (maintenance culture: 20-fold reduction; EPO: 18-fold reduction; G-/GM-CSF: 13-fold reduction - from day 0 to day 9). In acute leukemia, we found that miR-196a-1 was significantly higher expressed in AML compared to bone marrow samples of healthy donors (P=0.02). In T-ALL, miR-196a-1 was significantly up-regulated in patients with aberrant expression of myeloid markers (P=0.04), and miR-196b expression correlated with CD34 expression (P=0.003). In contrast to the reported adverse prognostic impact of ERG, expression of these miRNAs had no prognostic significance in T-ALL. CONCLUSION: This study identifies miR196a-1 and miR-196b as ERG regulators. We show that miR-196b is specifically down-regulated during hematopoietic differentiation, thus indicating a specific role of this miRNA in hematopoiesis. Moreover, the aberrant expression of miR-196a-1 and miR-196b in T-ALL and AML points to a potential role of these miRNAs in acute leukemias. Disclosures No relevant conflicts of interest to declare.

Introduction: Over the last years, genome and exome sequencing approaches have increased our knowledge of molecular alterations in acute myeloid leukemia (AML). However, some important limitations still need to be addressed. First, insights into the spectrum of molecular alterations of patients with refractory AML are rare, partly due to the lack of remission samples as germline control. As these patients have a dismal prognosis, there remains an unmet need to improve therapeutic options and to identify druggable molecular lesions. Secondly, in AML patients achieving a complete remission (CR), preleukemic alterations may persist in CR and are underestimated in frequency and relevance. In this work, we investigated mesenchymal stromal cells (MSC) as germline control to decipher the spectrum of molecular alterations in refractory patients with induction failure and to disclose preleukemic hits in patients achieving CR. Patients and methods : Bone marrow (BM) aspirates at initial diagnosis (ID) were obtained from 18 AML patients (9 pts with subsequent induction failure and 9 pts that achieved CR after first induction). MSC were expanded to passage 4 and defined as CD73+/CD105+/CD271+/low/CD45-/CD33- plastic-adherent cells. For all patients, BM hematopoietic cells (BM-HC; n=18) as well as MSC (n=18) were analysed at the time of first diagnosis. All samples (n=45) were analysed by exome sequencing on a HiSeq2500 (100 bp paired end) with four samples per lane. For variant calling, MSC were used as germline control. We demanded a variant allele frequency (VAF) of >20%, coverage of >30 reads and translational consequences. In germline samples, the VAF had to be < 5%. For patients achieving CR, BM-HC at CR were also studied (n=9). We repeated the analysis with CR BM-HC as germline control and compared the two results. For all patients, clinical as well as molecular characteristics were available. Results: We obtained an average coverage of 96 reads per base for the protein coding regions. 96% of the target region was covered at least 10-fold. The use of MSC as germline control allowed us to detect somatic mutations at initial diagnosis of refractory AML. In 9 refractory AML samples, we found 90 single nucleotide variants (SNV) and indels, which resulted in a median of 11 alterations per sample (range: 3-17). The spectrum of mutations showed an unexpectedly high rate of mutations in the spliceosome gene SRSF2 (3/9). Other recurrent mutations affected TET2 (2/9) and WT1 (2/9). Genes frequently mutated in non-selected AML were only present in one refractory patient (DNMT3A, RUNX1, IDH2, ASXL1, TP53, NRAS) or not found mutated (IDH1, KRAS). To uncover preleukemic alterations in AML patients achieving CR (n=9), we compared MSC and BM-HC at CR as germline controls. Using MSC as germline, we called 97 SNVs and indels (median: 11 per sample; range: 4-18) in the leukemic cells at ID. Thirty-three additional SNVs were called in the leukemic BM by using MSC as germline, whereas these would have been missed using BM-HC at CR as germline (median: 3 SNVs per sample, range: 0-7). These represent preleukemic hits persistent in CR with a VAF between 5% (lower bound) and 75%. Recurrently mutated genes included genes recently associated with clonal haematopoiesis in the elderly population: DNMT3A (3/9; VAF: 18%, 24%, 75%) and TET2 (2/9; VAF: 13%, 23%). In addition, mutations in ASXL1 (VAF: 14%), SRSF2 (VAF: 15%), and RUNX1 (VAF: 5%) persisted in at least one patient in CR. This unbiased approach also allowed us to identify lesions, which have not yet been associated with AML, but account for clonal events in remission. Candidates included genes linked to cancer like PROX1 (VAF: 5%), or ERBB2 (VAF: 35%), but also genes involved in NF-kB activation such as CARD8 (VAF: 30%), or NLRC3 (VAF: 10%). Conclusion: The use of MSC allows to unravel molecular lesions in refractory AML by exome sequencing. Refractory AML patients showed a high rate of mutations in the spliceosome gene SRSF2 that needs further investigations as potential therapeutic target for patients with treatment failure. Moreover, the comparison of two different germline controls (MSC and BM-HC in CR) allowed detecting persistent preleukemic alterations. In addition to known hits like in DNMT3A, TET2, or ASXL1, we systematically identified a broader spectrum of premalignant events that indicate clonal hematopoietic expansion and thereby may provide insights into leukemic transformation. Disclosures No relevant conflicts of interest to declare.