ALBERT

Description: Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of defective hematopoiesis of white blood cells and often characterized by limited treatment options and a poor prognosis. MicroRNAs, a class of small non-coding RNAs, have been reported over the last years to play an important role in the regulation of “normal” and pathological processes. By binding to the 3`UTR of their target mRNAs, microRNAs lead to the translational repression of their target gene. Deregulation of a certain microRNA thereby may lead to disrupted signal pathways, such as MAPK-signaling, and to tumorigenesis. However, the role of microRNAs in hematopoietic differentiation and blood cancer development remains largely unknown. In AML only few miRNAs are functionally characterized, and the clinical relevance of these miRNAs still remains to be determined. In this study we identified microRNA-143 (miR-143) to be highly upregulated in G-CSF stimulated CD34+ cells. We could also show the granulopoietic association of miR-143 in several hematopoietic cell line models. In addition, we show that miR-143 is upregulated in APL patients after ATRA treatment. QPCR analysis of pri-miR-143 expression in RIC (reduced intensity conditioning) transplanted AML patients reveal that high expression of miR-143 is associated with high event-free and overall survival. In functional studies, stable overexpression of miR-143 in K562-C/EBPα-p42 cells enhances the C/EBPalpha induced granulocytic differentiation significantly. In contrast, stable knock down of miR-143 reduced the ability of K562-C/EBPα-p42 cells to undergo granulocytic differentiation upon β-estradiol treatment. Moreover, chromatinimmunoprecipitation (ChIP) analysis show the direct binding of C/EBPα to the promoter region of miR-143. By in silico prediction, we found MAPK protein family members (MAPK1, MAPK3 and MAPK7) as predicted targets of miR-143. Western blot analysis of AML patient samples and G-CSF stimulated CD34+ cells clearly show an inverse correlation of miR-143 and MAPK7 (ERK5) protein expression. By transient overexpression of miR-143 we could show a strong downregulation of MAPK protein family member expression in NB4 cells. These studies suggest miR-143 as an essential factor in granulocytic differentiation driven by G-CSF. Deregulation of miR-143 may play an important role in the development of AML. Furthermore, we propose miR-143 as prognostic marker for AML patients undergoing RIC transplantation therapy. Disclosures No relevant conflicts of interest to declare.

Description: Key Points miR-30c is a direct target of C/EBPα and upregulated by C/EBPα-p42. NOTCH1 is a direct target of miR-30c and regulated by C/EBPα and miR-30c.

Description: The transcription factor CCAAT enhancer binding protein alpha (C/EBPα) is a master regulator of granulopoiesis and is silenced in approximately 50% of all acute myeloid leukemia (AML) cases. There are several mechanisms known how C/EBPα is inactivated in AML, including promoter hypermethylation, posttranslational modifications and mutations in the ORF of the CEBPA gene. MicroRNAs, a class of small non-coding RNAs, were identified as important regulators of normal hematopoiesis and leukemia development. We have already shown that microRNAs, such as miR-223, miR-34a and miR-30c, are essential elements in C/EBPα triggered granulocytic differentiation. But to our knowledge nothing is known about inactivation of C/EBPα by microRNAs in acute myeloid leukemia. In this study, we identified a novel network between C/EBPα and miR-182. In a next generation sequencing approach based on inducible K562-C/EBPα-ER cell line, we found miR-182 strongly downregulated by wildtype C/EBPα. We could further demonstrate an inverse correlation between C/EBPα protein amount and miR-182 expression level in several in vitro systems, including leukemic cell lines and G-CSF treated primary human CD34+progenitor cells. Additionally, C/EBPα and miR-182 showed reciprocal expression in sorted murine bone marrow subpopulations in vivo. To discover the mechanism how miR-182 is blocked by C/EBPα, we analyzed the minimal promoter region of miR-182 and performed chromatin immunoprecipitation (ChIP). Here, we could demonstrate a strong binding of C/EBPα to the miR-182 promoter, particularly to a conserved E2F binding site. Because E2F is a well known inhibitor of C/EBPα function, we tested whether E2F also effects miR-182 expression. An overexpression of E2F1 in U937 cells leads to an elevated miR-182 expression level. In addition, we measured the expression of miR-182 in bone marrow from AML patients regarding to their CEBPA mutation status. We could show that only patients with mutations in the C-terminal region of C/EBPα showed elevated miR-182 expression, while patients with N-terminal CEBPA mutations revealed no abnormal miR-182 expression compared to healthy donors or AML patients with no CEBPA mutation. The C-terminal domain of C/EBPα is necessary for E2F inhibition. These findings illustrate the importance of C/EBPα-E2F interaction during miR-182 regulation. Next, we found a highly conserved binding site of miR-182 in the 3’UTR of CEBPA itself, suggesting a possible negative feedback loop. To test this, we performed overexpression of miR-182 in U937 cells, umbilical cord blood mononuclear cells (UCB-MNCs) and primary blasts from AML patients. Here, we observed a strong reduction of C/EBPα protein after miR-182 overexpression in all cell types. Furthermore, we could demonstrate a direct binding of miR-182 to the 3’UTR of CEBPA via luciferase activity assay. Finally, we were interested in the functional impact of miR-182 in myeloid differentiation and leukemia development. We showed that enforced expression of miR-182 in U937 cells reduced the percentage of Mac-1 positive myeloid cells after treatment with all-trans retinoid acid (ATRA). Additionally, lentiviral overexpression of miR-182 induces a block of differentiation and hyperproliferation in G-CSF treated 32D cells and an enhanced replating capacity of primary mouse bone marrow mononuclear cells. Taken together, we identified miR-182 as novel oncogenic microRNA that directly blocks C/EBPα during myeloid differentiation and leukemia development. Thus, our data display a potential new strategy for therapeutics in C/EBPα dysregulated AML. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1361 In the acute promyelocytic leukemia (APL) bearing the translocation t(15;17), all-trans-retinoic acid (ATRA) treatment induces granulocytic maturation and complete remission of leukemia. Several factors are involved in the formation of the leukemic phenotype. Latest studies identified microRNAs as critical players in this network. In a micro array based microRNA screen we could identify miR-181b as downregulated in the APL cell line NB4 by treatment with pharmacological doses of ATRA. Additionally, we showed the transcriptional induction of miR-181b by the APL-associated PML-RARα oncogene which is released after treatment with ATRA. The overexpression of miR-181b by microRNA mimics leads to an inhibition of ATRA induced granulocytic differentiation. Furthermore, the downregulation of miR-181b by locked nucleic acids (LNAs) causes the induction of granulocytic transcription factor C/EBPβ. In a PML/RARα knock in mouse model we could show the enhanced expression of miR-181b. Also, APL patient samples show a significant enhanced expression of miR-181b in comparison to AML patient samples with normal karyotype. In addition, the miR-181b expression was strongly reduced in APL patient samples after ATRA treatment. In a microRNA target search we identified the novel ATRA regulated tumor suppressor RASSF1A as a putative target of miR-181b. In functional studies we showed that enforced expression of miR-181b reduces the protein level of RASSF1A by binding to the 3'UTR of RASSF1A mRNA. Accordingly, RASSF1A protein was enriched after knock down of miR-181b. The role of RASSF1A in ATRA induced differentiation was verified by knock down of RASSF1A protein by specific siRNA: Here we could show the reduction of ATRA induced CD11b expression. In conclusion, we identified miR-181b as a new player in the PML/RARα associated APL. Moreover, we firstly described the miR-181b target RASSF1A as a crucial factor in the ATRA activated granulocytic differentiation program in APL. Our data reveal the importance of deregulated miRNA biogenesis in cancer and may provide novel biomarkers and therapeutic targets in myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3516 Mitogen-activated protein kinase (MAPK) pathways are a family of related and sometimes interconnected pathways and one of the most studied. Over the last years, extensive work has established that these proteins play a critical role in G-CSF mediated maturation of neutrophil granulocytes. Understanding the mechanisms by which the MAPK pathways are regulated represents an important area of investigation. MicroRNAs, a class of small non-coding RNAs, have been found to play an important role in the regulation of diverse cellular processes by binding to target mRNAs leading to their translational repression. Deregulation of certain microRNAs, thereby, may lead to disrupted signal pathways, such as MAPK-signaling, and to tumorigenesis. However, the role of microRNAs in hematopoietic differentiation and development of leukemia remains largely unknown. In this study we performed a global screen to identify microRNAs involved in G-CSF-regulated MAPK-pathways in primary human CD34+ hematopoietic progenitor cells. Here we found microRNA-143 (miR-143) to be frequently upregulated in G-CSF stimulated CD34+ cells with a strong correlation to CD15 expression. We could also show the granulopoietic association of miR-143 in several hematopoietic cell line models and acute myeloid leukemia (AML) patient samples. Especially, AML patient samples FAB M4 and M5, which show monocytic phenotypes, had a significant lower expression level of miR-143 compared to the AML FAB types M0, M1, M2, and M3. In general, miR-143 expression was shown to be downregulated in AML patient samples in comparison to normal CD34+ hematopoietic progenitor cells. Most interestingly, we show that miR-143 expression is upregulated in acute promyelocytic leukemia (APL) patients after ATRA treatment. By in silico prediction we found MAPK protein family members (eg. MAPK1, MAPK3 and MAPK7) as predicted targets of miR-143. Western blot analysis of AML patient samples and G-CSF stimulated CD34+ cells clearly show an inverse correlation of miR-143 and MAPK7 (ERK5) protein expression. Finally, by transient overexpression of miR-143 we could show a strong downregulation of ERK protein expression in NB4 cells. Our study suggest that miR-143 upregulation by G-CSF may be an important regulatory step for permitting neutrophil differentiation. MicroRNA-143 blocks ERK5 signaling in G-CSF-induced granulopoiesis of CD34+ hematopoietic stem cells, is downregulated in myelo-monocytic acute myeloid leukemia subtypes, and upregulated after ATRA treatment in APL patients. This information may prove useful for the understanding of conditions in which neutrophil proliferation/differentiation balancing is dysregulated, such as in myeloid leukemia and myelodysplastic disorders. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3515 Almost 30% of all acute myeloid leukemias (AML) are associated with an internal tandem duplication (ITD) in the juxtamembrane of FLT3. This mutation leads to constitutive activated FLT3 signalling, which also includes altered FLT3 targets like STAT5. The dysregulation of pathways causes a differentiation block and plays a role in inhibition of hematopoietic transcription factors like PU.1 and C/EBPalpha. MicroRNA (miRNA) expression is also targeted by FLT3-ITD signaling. MiRNAs are small (19 – 22 nt) non coding RNAs, which regulate protein expression by binding to the 3'UTR of their target mRNAs. MicroRNA-155 (miR-155) was found to be highly expressed in FLT3-ITD associated AML patient samples. Thus, we hypothesized that miR-155 is an oncomiR which is regulated by FLT3-ITD signalling. Here we report that FLT3-ITD signaling induces the oncogenic miR-155 by NF-κB and STAT5 pathways. Furthermore we demonstrate that miR-155 targets the myeloid transcription factor PU.1. Analysis of FLT3-ITD positive patient samples show an approximately 2 fold higher miR-155 expression compared to FLT3-WT or FLT3-TKD associated AMLs. In addition we compared the miR-155 expression between 32D cells stably expressing either FLT3-WT or FLT3-ITD. The data show, that miR-155 expression is approximately 10 fold higher in the FLT3-ITD expressing 32D cells. Besides, the overexpression of FLT3-ITD in myeloid U937 cells increases miR-155 expression 2,2 fold. In contrast, a block of FLT3-ITD signalling in FLT3-ITD associated cell line MV4;11 by protein kinase inhibitors (PKIs), PKC412, SU5614 and CEP701, results in an 80% decreased miR-155 expression. In further experiments, we analyzed the role of FLT3-ITD downstream targets STAT5 and NF-κB (p65) in miR-155 regulation. We show that siRNA mediated knockdown of STAT5 or NF-κB (p65) in MV4;11 cells correlates with reduced miR-155 expression. A knockdown of NF-κB (p65) and STAT5 is able to overcome the FLT3-ITD overexpression induced miR-155 expression in U937 cells. To investigate the STAT5 impact on miR-155 expression in FLT3-ITD associated AML in vivo, we analyzed the miR-155 expression of irradiated C57BL/6 mice, transplanted with bone marrow from MX-Cre STAT5flox/flox mice stably expressing FLT3-ITD, after induced STAT5 knockout. The expression of miR-155 was significantly decreased to 0,4 fold in the STAT5−/− mice compared to STAT5flox/flox. These data reveal that STAT5 plays a pivotal role in FLT3-ITD induced miR-155 expression. In addition we demonstrate that p65 binds to the miR-155 promoter in MV4;11 cells while the treatment with the PKI CEP701 results in a p65 dissociation from the miR-155 promoter. Furthermore, we prove that miR-155 overexpression in PMA (phorbol 12-myristate 13-acetate) treated U937 cells reduces macrophage differentiation about 50%. In in silico analysis we found PU.1 as a putative miR-155 target. Interestingly, we could show that overexpression of FLT3-ITD as well as overexpression of miR-155 reduces the PU.1 protein level in U937 cells to 0.5 fold. However, a block of miR-155 by locked nucleic acids (LNAs) in MV4;11 cells, leads to PU.1 protein increase. Further, by luciferase assay analyses we prove that miR-155 directly targets PU.1 3'UTR. Altogether we conclude that FLT3-ITD induces the oncogenic miR-155 via STAT5 and NF-κB (p65). The increased miR-155 expression targets PU.1 and enhances the myeloproliferative disorder in acute myeloid leukemia blasts. These data reveal the impact of deregulated miRNAs in cancer development and may provide novel biomarkers and therapeutic targets in leukemia. Disclosures: No relevant conflicts of interest to declare.

Description: In acute promyelocytic leukemia (APL) bearing the translocation t(15;17), all-trans-retinoic acid (ATRA) treatment induces granulocytic maturation and complete remission of leukemia. Several factors are involved in the formation of the leukemic phenotype. Latest studies identified microRNAs as critical players in this network. In a micro array based microRNA screen we could identify the genomically clustered miR-181a and miR-181b as downregulated in the APL cell line NB4 by treatment with pharmacological doses of ATRA. In addition, the expression of the miR-181a/b-cluster was strongly reduced in bone marrow samples of APL patient while ATRA-based therapy. Furthermore, we showed the transcriptional induction of miR-181a and miR-181b by the APL-associated PML-RARα oncogene in vitro and in vivo. In PR9 cells, carrying a zinc-driven PML/RARα construct, and in PML/RARα-knock in mice the expression of the fusion gene leads to upregulation of the microRNA-cluster expression. Analysis of bone marrow samples of APL patients showed an enhanced expression of miR-181a and miR-181b in comparison to AML patient samples with normal karyotype, whereas other AML subgroups show no significant regulation. Based on siRNA experiments we could propose AP-1 and GATA-2 as potential co-activators for the PML/RARα-dependent regulation of the miR-181a/b-cluster. In functional studies in NB4 cells we observed after lentiviral knock down of miR-181a and miR-181b a significant reduction of colony size and number as well as proliferation rate. In contrast, transient overexpression of miR-181a and miR-181b led to an inhibition of ATRA-induced expression of the differentiation marker CD11b. In a microRNA target search we identified the novel ATRA regulated tumor suppressor RASSF1A as a putative target of miR-181a and miR-181b. In functional studies we showed that enforced expression of miR-181a and miR-181b reduces the protein level of RASSF1A by binding to the 3´UTR of RASSF1A mRNA. Accordingly, RASSF1A protein was enriched after knock down of miR-181b. The role of RASSF1A in ATRA induced differentiation was verified by knock down of RASSF1A protein by specific siRNA: Here we could show the reduction of ATRA induced CD11b expression. Overexpression of RASSF1A in NB4 cells strongly induced apoptosis. Additional, we could show by western blot that the miR-181a/b-cluster and RASSF1A modulate cell cycle via regulation of cyclin D1. In conclusion, we identified the miR-181a/b-cluster as an important player in the PML/RARα associated APL. Moreover, we firstly described the miR-181a/b target RASSF1A as a crucial factor in the ATRA activated granulocytic differentiation program in APL. Our data reveal the importance of deregulated microRNA biogenesis in cancer and may provide novel biomarkers and therapeutic targets in myeloid leukemia. Disclosures No relevant conflicts of interest to declare.