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DNMT1 protein expression in SKM1 AZA-sensitive (-S) versus AZA-resistant clones (-R). The Figure corresponds to the Figure 1B with signal quantification performed with ImageJ software. The data are expressed as mean {plus minus} SD of three independent experiments, with each performed in triplicate (Mann-Whitney test, p

Pathway enrichment analysis. For ontology analysis, gene lists were analyzed using DAVID software (KEGG pathways). The complete set of genes featured in the microarrays was used as the reference background. The three numbers on the right represent the number of deregulated mRNAs, the overall number of genes within the pathway and the p value, respectively. Data are presented for genes targeted by at least one of the 7 miRNAs and that were found repressed in SKM1 AZA resistant cells.

Purpose: Azacitidine inhibits DNA methyltransferases, including DNMT1, and is currently the standard of care for patients with higher-risk myelodysplastic syndrome (HRMDS) or low blast count acute myeloid leukemia (AML).Experimental Design: The expression of 754 miRNAs was compared in azacitidine-resistant and azacitidine-sensitive myelodysplastic syndrome cells. We investigated the role of differentially expressed miRNAs on DNMT1 expression and azacitidine resistance in vitro. We next evaluated anti-DNMT1 miRNA expression in pretreatment bone marrow samples derived from 75 patients treated with azacitidine for HRMDS or AML.Results: Seven miRNAs, including 5 that in silico targeted the DNMT1 3′ UTR, were repressed in azacitidine-resistant cells in which DNMT1 protein levels were significantly higher. Ectopic anti-DNMT1 miRNA expression decreased DNMT1 expression and increased azacitidine sensitivity, whereas specific inhibition of endogenous anti-DNMT1 miRNAs increased DNMT1 expression and triggered azacitidine resistance. In patients treated with azacitidine, decreased expression of anti-DNMT1 miRNAs was associated with poor outcome. miR-126* had the strongest prognostic impact. Patients with miR-126*low myelodysplastic syndrome had significantly lower response rates (P = 0.04) and higher relapse rates (P = 0.03), as well as shorter progression-free (PFS; P = 0.004) and overall survival (OS; P = 0.004). Multivariate analysis showed that age, miR-126* expression, and revised International Prognostic Scoring System risk independently predicted PFS and OS. In 15 patient samples collected over time, decreased miRNA expression levels were associated with secondary resistance.Conclusions: A decreased expression of anti-DNMT1 miRNAs might account for azacitidine resistance in HRMDS and AML, and measuring miRNA expression before and during treatment might help predict primary or secondary azacitidine resistance. Clin Cancer Res; 23(12); 3025–34. ©2016 AACR.

Acute Myeloid Leukemia (AML) is characterized by the expansion and resistance to apoptosis of myeloid cells blocked in early stages of differentiation. A subset of stem or progenitor cells, termed leukemic stem cells (LSC), which retain or reacquire self-renewing properties, as well as the capacity to remain in a poorly differentiated stage, give rise to the leukemic clone. The identification and purification of the LSC can provide a powerful tool for diagnosis, prognosis and therapy. Several studies suggested that LSC belong to the CD34+ CD38- compartment. Self-renewal and lack of differentiation are properties of embryonic and induced pluripotent stem cell which specifically express a set of differentiation antigens (SSEA1 and SSEA3) and transcription factors (OCT¾, SOX2, and NANOG), designated as ESCA. We postulate that an epigenetic reprogramming could induce the ESCA expression on HSC. The aim of our study was to investigate the expression ESCA in the CD34+CD38- cells from normal bone marrow (NBM) and from 50 AML BM patients. Then compare ESCA's expression between CD34+CD38- and CD34+CD38+ cells. We studied the expression in 5 cell lines (NTERA-2 which is control cell line, KG1a, U937, THP-1 and HL60 leukemic cell lines) and their potential involvement in myeloid differentiation in 2 cell lines (HL60 and THP-1). Thereafter, we inhibited the expression of ESCA in 3 leukemic cell lines (HL60, KG1a and U937) to better understand their role in abnormal proliferation or differentiation. The preliminary experiments showed an important expression of ESCA on all 5 cell lines by Multicolor Flow Cytometry (MFC), confirmed by RT-PCR. Then, we compared the expression of ESCA in CD34+CD38- cells between normal and leukemic marrow. We observed an up-regulation of two transcription factors OCT3/4 and SOX2 and the protein SSEA3 with 2-fold higher expression in AML cells compared to normal HSC. In addition, we found the down regulation of SSEA1 protein involved in cell adhesion, migration and differentiation. We also compared the expression of ESCA between the CD34+CD38- and CD34+CD38+ AML population. We observed a higher expression of OCT3/4 and SSEA3 (1.3-fold) in CD34+CD38-. We found a higher expression of SSEA1 (1.9-fold), NANOG and SOX2 (1.2-fold) in AML CD34+CD38+ population compared to CD34+CD38- population. We investigated whether there was a relationship between the overexpression of ESCA and a cytogenetic subgroup of AML. We found that AML t(15;17) expressed OCT3/4 at higher level than the other ESCA. Finally, the inhibition experiments showed that Oct3/4 was strongly inhibited in the KG1a cell line. In conclusion, these results suggest that the deregulation of ESCA may have a potential role in leukemogenesis by maintaining the LSC properties. This prompts us to test the relevance of these markers for LSC identification and as therapeutic strategies. Citation Format: Lydia Campos, TIPHANIE PICOT, CARMEN AANEI, PASCALE FLANDRIN-GRESTA, EMMANUELLE TAVERNIER, DENIS GUYOTAT. Embryonic stem cells antigens: expression in acute myeloid leukemia cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1961. doi:10.1158/1538-7445.AM2014-1961

Objectives: The recent evidences demonstrate that in myelodysplastic syndromes (MDS) a particular role is played by stromal microenvironment dysfunctions, which mediate the direct contact with hematopoietic precursor cells (HPC). The aims of our study were to assess the putative growth deficiencies of mesenchymal stromal cells (MSC) selected from MDS individuals, with regard to their ability to generate a microenvironment suitable to HPC development. Then, we intended to decode the focal adhesion (FA) signalling pathways and to understand whether adhesion-mediated processes contribute to transduction of intrinsic proliferative signals, as well as their impact on HPC-to-MSC interactions. Methods: To this end, we imagined a selection procedure using MSC specific markers expression (STRO-1 and CD73), we performed their phenotypic evaluation, and we conducted the functional assays on MSC and HPC selected from MDS patients vs. healthy volunteers. Finally, we have used the immunofluorescence microscopy to characterize the FA proteins (paxillin and focal adhesion kinase [FAK]), and of their regulators, p130CAS and HSP90. Results: The MSC production in STRO-1+ and CD73+ cell cultures from refractory cytopenia (RC) marrows was deficient, and, in addition, the clonogenic ability of these fractions was strongly diminished. The relative proliferation in MSC cultures from RC is the result of a continuous division process occurring at a low rate and lacking the ability to generate the normal functional progenitors required to form colonies. By contrast, in refractory anemia with excess of blasts (RAEB) settings, the proliferation rate is moderately improved due to the reduced doubling time of STRO-1 cells. However, this was not accompanied, at the end point, by complete functional maturity as reflected in the CFU-F number. Likewise, we have to point out the diminution of CFU-F capacity of CD73+ fractions in MDS that directly correlates with the CD44 mitigate on their surface. In addition, the doubling time of MSC from MDS inversely correlate with their expression for CD49e (Δ5-integrin). The proliferation differences occur in MDS cultures compared to normal settings can be attributed equally to the qualitative defects of FA proteins (FAK, and paxillin). The MSC from RAEB cultures highlight a strong complexation of FA proteins to HSP90 in nuclear area, which support a proliferative behaviour of these cells. Moreover, this high colocalisation to HSP90 indicates the cessation of proteasome-mediated recycling of these proteins. Furthermore, the preliminary results indicate the fact that the clonogenic potential of HPC is controlled by adhesion mechanisms dependent on stroma, and FAK is one of the molecules involved in this process. Conclusions: These data prove that MSC selected from MDS patients are intrinsically pathological and they could influence HSC behaviour by their direct interactions via FA proteins signalling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2470. doi:1538-7445.AM2012-2470