Mir-199b, a Novel Tumor Suppressor miRNA In Acute Myeloid Leukemia With Prognostic Implications In FAB-M5 Subtype

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1271-1271
Author(s):  
Amanda J. Favreau ◽  
Christine W. Duarte ◽  
Pradeep Sathyanarayana
Keyword(s):  
Acute Myeloid Leukemia ◽  
Mouse Model ◽  
Mirna Expression ◽  
Myeloid Leukemia ◽  
Hdac Inhibitors ◽  
Cd34 Cells ◽  
Prognostic Implications ◽  
Low Expression ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) manifests a marked heterogeneity in both response to therapy and patient survival, observations that likely reflect its varied pathogenesis. Dysregulation of miRNAs that can act as tumor suppressors or oncogenes can result in tumorigenesis. One miRNA of interest is human miR-199b-5p, which is an intragenic miRNA encoded in the Dynamin 1 gene. Previously we demonstrated that miR-199b was significantly downregulated in AML and targets Podocalyxin and Discoidin Domain Receptor 1. Herein we investigated the functional role of miR-199b in AML and its prognostic implications. We first examined the relative miR-199b expression in steady state hematopoiesis and showed CD33+ myeloid progenitors had the highest miR-199b expression (p<0.03). In order to test whether suppression of miR-199b results in myeloproliferation, human bone marrow derived CD34+ cells were transduced with anti-miR-199b or control lentivirus particles. To examine the effect of miR-199b silencing on colony forming abilities, a CFU assay was performed. At day 16, silencing of miR-199b in CD34+ cells resulted in significant increases in CFU-GM colonies. We then investigated the effect of decreased miR-199b on hematopoietic stem cells (HSC) proliferation via aldehyde dehydrogenase assay. Anti-miR-199b CD34+ cells exhibited a significant increase in HSC numbers compared to control CD34+ cells demonstrating a proliferative advantage for HSC with decreased miR-199b. Via The Cancer Genome Atlas (TCGA), we analyzed the molecular and clinical characteristics of 166 AML cases to investigate a prognostic role for miR-199b. The Kaplan-Meier curves for high and low expression values of miR-199b and the observed distribution of miRNA expression revealed the highly expressed group had significantly better survival outcomes (p<0.016, log rank test). In addition, miR-199b expression varied by cytogenetic risk category in the least squared mean plot, with significantly higher miR-199b expression for the favorable category compared to the intermediate and poor categories combined (p<0.0001). Therefore, low expression of miR-199b predicts worse survival outcome and higher cytogenetic risk in AML. Additionally, there was significant difference between miR-199b expression across the AML subtypes with particularly low expression found in the M5 subtype. Furthermore, M5 subtype showed a poor prognosis with a one-year survival rate of only 25%, compared with 51% survival in the overall sample (p<0.024). With AML patients grouped into M5 versus other subtypes to look at the dichotomized miR-199b variable, we found that all of the M5 patients have low miR-199b expression (p<0.0001). Due to miR-199b significantly correlating with M5 subtype, the M5 cell line THP-1 was utilized to determine the epigenetic regulation of miR-199b in AML. Recently, epigenetic alterations such as histone modifications and DNA methylation have been shown to deregulate miRNA expression. Therefore, treatment of THP-1 cells for 24 hours with HDAC inhibitors AR-42 (2uM), Panobinostat (0.7uM), or Decitabine (demethylating agent, 5uM) showed miR-199b expression was significantly elevated upon AR-42 and Panobinostat treatment (p<0.0002 and p<0.0001 respectively). Furthermore, AR-42 and Panobinostat treated cells showed drastic apoptosis in both treatments. Thus, epigenetic regulation of miR-199b appears to be via histone modification. To further understand the hematopathological consequences of decreased miR-199b in the onset and development of myeloid leukemia, we employed a transduce / transplant mouse model. The Lin-Sca-1+Kit+ (LSK) population was isolated from Ly5.2 C57Bl6 mice, transduced with anti-miR-199b or control lentivirus particles, and transplanted into Ly5.1 C57Bl6 recipient mice. Short term (3.5 weeks) peripheral blood analysis revealed that loss of miR-199b results in significant increase of % neutrophils (p<0.001), % monocytes (p<0.04), and % eosinophils (p<0.02). Further analysis to determine the role of miR-199b in leukemogenesis is ongoing via this mouse model. Taken together, for the first time our results demonstrate that in vivo loss of miR-199b can lead to myeloproliferation and in vitro HDAC inhibitors can restore miR-199b-5p expression and promote apoptosis. Lastly, low miR-199b-5p in AML patients leads to worst overall survival and has prognostic significance for FAB-M5 subtype. Disclosures: No relevant conflicts of interest to declare.

MicroRNA-29a Is An Oncogene in Acute Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 683-683
Author(s):  
Christopher Y. Park ◽  
Yoon-Chi Han ◽  
Govind Bhagat ◽  
Jian-Bing Fan ◽  
Irving L Weissman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Hematopoietic Stem Cells ◽  
Mirna Expression ◽  
Myeloid Leukemia ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract microRNAs (miRNAs) are short, non-protein encoding RNAs that bind to the 3′UTR’s of target mRNAs and negatively regulate gene expression by facilitating mRNA degradation or translational inhibition. Aberrant miRNA expression is well-documented in both solid and hematopoietic malignancies, and a number of recent miRNA profiling studies have identified miRNAs associated with specific human acute myeloid leukemia (AML) cytogenetic groups as well as miRNAs that may prognosticate clinical outcomes in AML patients. Unfortunately, these studies do not directly address the functional role of miRNAs in AML. In fact, there is no direct functional evidence that miRNAs are required for AML development or maintenance. Herein, we report on our recent efforts to elucidate the role of miRNAs in AML stem cells. miRNA expression profiling of AML stem cells and their normal counterparts, hematopoietic stem cells (HSC) and committed progenitors, reveals that miR-29a is highly expressed in human hematopoietic stem cells (HSC) and human AML relative to normal committed progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors is sufficient to induce a myeloproliferative disorder (MPD) that progresses to AML. During the MPD phase of the disease, miR-29a alters the composition of committed myeloid progenitors, significantly expedites cell cycle progression, and promotes proliferation of hematopoietic progenitors at the level of the multipotent progenitor (MPP). These changes are manifested pathologically by marked granulocytic and megakaryocytic hyperplasia with hepatosplenomegaly. Mice with miR-29a-induced MPD uniformly progress to an AML that contains a leukemia stem cell (LSC) population that can serially transplant disease with as few as 20 purified LSC. Gene expression analysis reveals multiple tumor suppressors and cell cycle regulators downregulated in miR-29a expressing cells compared to wild type. We have demonstrated that one of these genes, Hbp1, is a bona fide miR-29a target, but knockdown of Hbp1 in vivo does not recapitulate the miR-29a phenotype. These data indicate that additional genes are required for miR-29a’s leukemogenic activity. In summary, our data demonstrate that miR-29a regulates early events in normal hematopoiesis and promotes myeloid differentiation and expansion. Moreover, they establish that misexpression of a single miRNA is sufficient to drive leukemogenesis, suggesting that therapeutic targeting of miRNAs may be an effective means of treating myeloid leukemias.

scholarly journals HDAC8 Promotes MLL-Rearranged Acute Myeloid Leukemia By Inhibiting p53 Acetylation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 642-642
Author(s):  
Dijiong Wu ◽  
Man Li ◽  
Lianjun Zhang ◽  
Jing Qi ◽  
Chun-Wei David Chen ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Median Survival ◽  
Myeloid Leukemia ◽  
Fold Change ◽  
Poly I:C ◽  
White Blood Count ◽  
Cd34 Cells ◽  
Acute Myeloid

Chromosomal rearrangements involving the mixed lineage leukemia (MLL) gene at the 11q23 locus are found in a subset of acute myeloid leukemia (AML) generally associated with intermediate to poor prognosis. MLL-AF9 resulting from t(9;11)(p22;q23) is the most common in MLL-rearranged (MLL-r) AML. Chemotherapy is the current standard of care for AML and the overall 5-year survival rate is only 25%. We previously reported that HDAC8 functions to control p53 activity in hematopoietic stem cells via deacetylation of p53 protein (Hua et al, Blood 2017). Here, we investigated the contribution of HDAC8 in MLL-r AML pathogenesis and maintenance. We have previously generated a conditional Hdac8 floxed (f) allele and combined with Mx1-Cre (Mx1-Cre/Hdac8f/f(y)) to generate HDAC8△/△ mice upon induction with poly (I:C). To examine the role of HDAC8 in MLL-AF9-AML, we transduced Hdac8△/△ or control LSK (Lin-Sca1+cKit+) with a MSCV-ires-GFP (MIG)-MLL-AF9 (MA9) vector and sorted GFP+ cells for CFC assay. Hdac8△/△/MA9 cells generated significantly less CFC colonies compared to control, suggesting that HDAC8 promotes MA9 clonogenic activity. To assess their leukemogenic potential in vivo, we transplanted GFP+ control MA9 or Hdac8△/△/MA9 cells (5x103-104) into sublethally irradiated (6 Gy) wild-type (WT) recipients. All MA9 recipients (n=4) developed lethal AML with a median survival of 69 days while none of the Hdac8△/△/MA9 recipients (n=4) developed AML up to 4 months. To examine the contribution of HDAC8 in established AML, we transduced LSK from uninduced Hdac8f/f(y)/Mx1-Cre mice with MIG-MA9 and transplanted into WT recipients. Upon development of moribund leukemia, AML cells (5x 103) were transplanted into a cohort of recipients (n=16), which were treated with poly (I:C) (n=8; 14 mg/kg/dose) or PBS every other day for 2 weeks starting at day 10 (5-10% GFP+ in blood). Mice induced with poly (I:C) to delete Hdac8 showed dramatically lower white blood count [9.0 (103 /ul) vs. 109.7 (103 /ul); n=8, P&lt;0.05] and lower AML burden (GFP+) in peripheral blood (9.3% vs 42.3%; n=8, P&lt;0.05) at 3 weeks. Prolonged survival was observed in poly (I:C) treated mice compared to control (median survival 36 days vs 27 days, n=8, P&lt;0.01). These results indicate that HDAC8 deletion impairs AML progression. Thus, we examined the effects of HDAC8-selective inhibitor (HDAC8i) on MA9 AML cell proliferation and survival. Treatment of MA9 AML cells with HDAC8i (22d) led to dose-dependent inhibition of cell survival (IC50 = 4.724 - 8.453 mM) and increased apoptosis (10 mM, 48 h) compared to vehicle treated control (40% vs. 7%). HDAC8i treatment significantly enhanced p53 acetylation, and upregulated p53 target genes, Gadd45d (fold change = 3.2; p&lt; 0.01), Puma (fold change = 5.7; p&lt; 0.01), Mdm2 (fold change = 3.0; p&lt; 0.01) and Cdkn1a (fold change = 3.6; p&lt; 0.01). To assess the effects on AML growth and leukemia-initiating capacity in vivo, we transplanted MA9 AML cells treated with HDAC8i (1 x 105, 2.5x105) or vehicle (2.5x105) into sublethally irradiated WT recipients (n=8-11). We observed significantly (p&lt;0.0001) reduced GFP+ AML burden in recipients of HDAC8i-treated cells (both 1x105 & 2 x105) and significantly (p&lt;0.0001) improved survival. Secondary transplantation of AML cells from HDAC8i- or vehicle-treated (2 x105) recipients revealed significantly improved survival in HDAC8i-treated group (22d 105 days n=4 vs. vehicle 33 days n=5; p&lt; 0.01). These results indicate that HDAC8 inhibition enhanced elimination of MA9 AML cells and leukemia-initiating cells. We further evaluated the effect of HDAC8i in human AML cell lines and primary MLL-r AML patient cells. HDAC8i treatment increased p53 acetylation and enhanced p53-dependent apoptosis in MOLM-13 AML cells (22d: 50.8% vs. vehicle: 11.89%). Knock-down of p53 rescued the apoptosis induced by HDAC8i, indicating that HDAC8i induced apoptosis is mediated by p53. Similarly, HDAC8i (22d) treatment significantly increased apoptosis accompanied by elevated levels of p53 acetylation and p53 target gene expression in MLL-r AML CD34+ cells compared to normal (NL) CD34+ cells. Thus, HDAC8 inhibition targets human MLL-r AML cells and AML CD34+ cells by restoring p53 acetylation and inducing apoptosis. In conclusion, this study reveals a critical role of HDAC8 in promoting initiation and maintenance of MLL-AF9 AML and highlights HDAC8 inhibition as a promising approach to target MLL-r AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Acute Myeloid Leukemia Cell-Intrinsic PD-1 Functions Promoting Leukemia Development By Recruiting SHP2

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1173-1173
Author(s):  
Shanshan Suo ◽  
Le Xuan Truong Nguyen ◽  
Fenglin Li ◽  
Dandan Zhao ◽  
Junjing Qiao ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Cell Growth ◽  
Median Survival ◽  
Myeloid Leukemia ◽  
Programmed Death ◽  
Cd34 Cells ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a devastating hematopoietic malignancy. With current therapies, only approximately 30% of patients achieve long-term survival. Therefore, novel, more active and less toxic treatments are urgently needed. Programmed death-1 (PD-1) is a cell surface receptor that functions as a T cell checkpoint and plays a central role in regulating T cell exhaustion. Binding of PD-1 to its ligand, programmed death-ligand 1 (PD-L1), activates downstream signaling pathways and inhibits T cell activation. Abnormally high PD-L1 expression on tumor cells and antigen-presenting cells in the tumor microenvironment mediates tumor immune escape, and PD-1/PD-L1 immune checkpoint blockade has showed promising results in cancer patients. Recently, PD-1 expressed on melanoma cells was also shown to play a pivotal role in tumor growth. To date, in AML, the function of PD-1 has been mainly studied in the host T cells, while little is known regarding the role of PD-1 in AML cells. Herein, we examined the level and role of PD-1 in AML cells using AML murine model and patient samples. We used MLL PTD/WT/Flt3 ITD/ITD knock-in mouse in B6 background, a well characterized AML model, to study the expression and function of PD-1 in AML. Flow cytometric analysis of LSK (Lin -Sca-l1 +c-kit +) cells from the bone marrow (BM) of wild-type (WT, n=5) and AML (n=10) mice showed that 20.9%-61.9% of AML LSKs versus (vs) &lt;5.0% of normal LSKs are PD-1 positive (P&lt; 0.0001). Western blot and Q-RT-PCR analysis confirmed higher levels of PD-1 in AML LSKs than in normal LSKs. The PD-1 levels on LSKs increased over time and associated with disease progression. Similar results were obtained in AML patients, showing PD-1 + in 0.2%-14.6% of AML CD34 + cells vs &lt; 1.0% of normal CD34 + cells (P&lt; 0.05). Data analysis based on TCGA showed that higher PD-1 levels are associated with shorter survival in AML patients (P=0.0125). To assess the functional role of PD-1 in AML, we sorted PD-1 + and PD-1 - fractions fromAML LSKs and observed a lower frequency of quiescent cells (G0, 16.60% vs 44.87%, P&lt; 0.05) and a higher cell growth rate in the PD-1 + vs PD-1 - cells. Further in vivo study showed that PD-1 + AML LSKs (CD45.2) generated higher white blood cell (WBC) counts (P&lt; 0.0001), higher AML engraftment (P&lt; 0.0001) and a shorter survival (median survival 57.5 vs &gt;75 days, P&lt; 0.001) in recipient mice (CD45.1) compared with PD-1 - AML LSKs. Similar results were observed in human samples. Compared to PD-1 - CD34 + cells, PD-1 + CD34 + cells are less quiescent and more proliferative (P&lt; 0.01). PD-1 + AML blasts had higher engraftment rate (13.18% vs 2.68%, p=0.0002) and shorter survival (median survival: 27 vs 45 days, P= 0.0008) in NSGS mice than PD-1 - AML blasts. To evaluate if these in vivo differences observed in PD-1 + vs PD-1 - AML LSKs were mediated by interactions between PD-1 + AML and T cells, PD-1 + and PD-1 -LSKs from AML mouse were transplanted into T-, B- cell-deficient NSG mice. Recipient mice transplanted with PD-1 + AML LSKs had higher WBC counts (P&lt; 0.01), higher AML engraftment (P&lt; 0.0001) and a shorter survival (median survival: 76 vs &gt;130 days, P&lt; 0.0001) than recipients with PD-1 - AML LSKs, suggesting that these differences were T cell-independent. Next, we examined whether blocking PD-1 could affect leukemic cell growth. We sorted LSK cells from AML mice and performed colony forming cell (CFC) assay in the presence of anti-PD1 antibody or isotype antibody. Blocking PD-1 with anti-PD-1 antibody significantly suppressed CFC and cell growth in vitro but did not induce apoptosis compared to isotype control antibody. To explore the molecular mechanism by which PD-1 contributes to AML growth, we then sorted PD-1 + and PD-1 - LSKs from AML mice for molecular studies. Western blot assays revealed higher levels of SHP-2 and phosphorylated (p) -ERK in PD-1 + vs PD-1 - AML LSKs. We validated these results in primary human AML cells by immunofluorescence staining. Confocal microscopy of PD-1 + and PD-1 - human AML CD34 +cells demonstrated that PD-1 localized at the cell membrane and in the cytoplasm and p-ERK was markedly enhanced in the PD-1 + CD34 + cells. In conclusion, we showed here that a subpopulation of murine and human AML blasts expresses high levels of PD-1 which mediated disease initiation and growth through activation of the MAPK/ERK signaling pathways. PD-1 blocking antibody reversed these activities and might contribute to the clinical efficacy of anti-PD-1 therapy in AML. Disclosures Marcucci: Novartis: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings.

scholarly journals CGRP Signaling via CALCRL Increases Chemotherapy Resistance and Stem Cell Properties in Acute Myeloid Leukemia

2019 ◽  
Vol 20 (23) ◽  
pp. 5826 ◽  
Author(s):  
Tobias Gluexam ◽  
Alexander M. Grandits ◽  
Angela Schlerka ◽  
Chi Huu Nguyen ◽  
Julia Etzler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Mouse Model ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Acute Myeloid

The neuropeptide CGRP, acting through the G-protein coupled receptor CALCRL and its coreceptor RAMP1, plays a key role in migraines, which has led to the clinical development of several inhibitory compounds. Recently, high CALCRL expression has been shown to be associated with a poor prognosis in acute myeloid leukemia (AML). We investigate, therefore, the functional role of the CGRP-CALCRL axis in AML. To this end, in silico analyses, human AML cell lines, primary patient samples, and a C57BL/6-based mouse model of AML are used. We find that CALCRL is up-regulated at relapse of AML, in leukemic stem cells (LSCs) versus bulk leukemic cells, and in LSCs versus normal hematopoietic stem cells. CGRP protects receptor-positive AML cell lines and primary AML samples from apoptosis induced by cytostatic drugs used in AML therapy, and this effect is inhibited by specific antagonists. Furthermore, the CGRP antagonist olcegepant increases differentiation and reduces the leukemic burden as well as key stem cell properties in a mouse model of AML. These data provide a basis for further investigations into a possible role of CGRP-CALCRL inhibition in the therapy of AML.

scholarly journals Loss of erythroblasts in acute myeloid leukemia causes iron redistribution with clinical implications

2021 ◽  
Vol 5 (16) ◽  
pp. 3102-3112
Author(s):  
Marta Lopes ◽  
Tiago L. Duarte ◽  
Maria J. Teles ◽  
Laura Mosteo ◽  
Sérgio Chacim ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Mouse Model ◽  
Myeloid Leukemia ◽  
Transferrin Saturation ◽  
Treatment Strategies ◽  
Leukemic Cells ◽  
Cancer Types ◽  
Iron Profile ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease with poor prognosis and limited treatment strategies. Determining the role of cell-extrinsic regulators of leukemic cells is vital to gain clinical insights into the biology of AML. Iron is a key extrinsic regulator of cancer, but its systemic regulation remains poorly explored in AML. To address this question, we studied iron metabolism in patients with AML at diagnosis and explored the mechanisms involved using the syngeneic MLL-AF9–induced AML mouse model. We found that AML is a disorder with a unique iron profile, not associated with inflammation or transfusion, characterized by high ferritin, low transferrin, high transferrin saturation (TSAT), and high hepcidin. The increased TSAT in particular, contrasts with observations in other cancer types and in anemia of inflammation. Using the MLL-AF9 mouse model of AML, we demonstrated that the AML-induced loss of erythroblasts is responsible for iron redistribution and increased TSAT. We also show that AML progression is delayed in mouse models of systemic iron overload and that elevated TSAT at diagnosis is independently associated with increased overall survival in AML. We suggest that TSAT may be a relevant prognostic marker in AML.

scholarly journals Loss of erythroblasts in acute myeloid leukemia causes iron redistribution with clinical implications

2020 ◽  
Author(s):  
Marta Lopes ◽  
Tiago L. Duarte ◽  
Maria J. Teles ◽  
Laura Mosteo ◽  
Sérgio Chacim ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Mouse Model ◽  
Myeloid Leukemia ◽  
Transferrin Saturation ◽  
Treatment Strategies ◽  
Leukemic Cells ◽  
Cancer Types ◽  
Iron Profile ◽  
Acute Myeloid

AbstractAcute myeloid leukemia (AML) is a heterogeneous disease with poor prognosis and limited treatment strategies. Determining the role of cell-extrinsic regulators of leukemic cells is vital to gain clinical insights into the biology of AML. Iron is a key extrinsic regulator of cancer but its systemic regulation remains poorly explored in AML. To address this question, we studied iron metabolism in AML patients at diagnosis and mechanisms involved using the syngeneic MLL-AF9-induced AML mouse model. We found that AML is a disorder with a unique iron profile not associated with inflammation or transfusion and characterized by high ferritin, low transferrin, high transferrin saturation (TSAT), and high hepcidin. The increased TSAT in particular, contrasts with observations in other cancer types and in anemia of inflammation. Using the MLL-AF9 mouse model of AML, we demonstrated that leukemic blasts take up iron and that the AML-induced loss of erythroblasts is responsible for iron redistribution and an increase in TSAT. We also show that elevated TSAT at diagnosis is independently associated with increased overall survival in AML and suggest that TSAT may be a relevant prognostic marker in AML.

scholarly journals The Role of the Slit-Robo Family in Adult Patients with Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3816-3816
Author(s):  
Aleksandra Golos ◽  
Dorota Jesionek-Kupnicka ◽  
Tadeusz Robak ◽  
Ewa Wawrzyniak ◽  
Lidia Anna Gil ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Bone Marrow Involvement ◽  
Control Group ◽  
Newly Diagnosed ◽  
Low Expression ◽  
Acute Myeloid

Abstract Introduction: SLIT-ROBO is newly discovered ligand-receptor family of neuronal guidance molecules. Recently, it has been proved that these proteins are involved in both, physiologic and pathologic angiogenesis. In animal models, it was shown both, pro-and antiangiogenic of SLIT-ROBO signaling. Moreover, the interaction of SLIT ligands with their roundabout receptors (ROBO) results in promotion of apoptosis, adhesion and blocking of cell cycle. There is evidence that SLIT-ROBO proteins are involved in pathogenesis of solid tumors, both in angiogenesis dependent and independent way. The role of SLIT-ROBO proteins in biology of acute myeloid leukemia (AML) remains unknown.The only two hitherto published studies considering ROBO4 expression in AML have revealed its increased expression in the blasts cells. The aim of the study was to evaluate the role of SLIT-ROBO proteins in AML. The expression of SLIT ligands, and their receptors ROBO was assessed in bone marrow of newly diagnosed AML patients and in the control group. The expression level of the proteins was correlated with known prognostic factors, response to treatment and overall survival (OS), as well as angiogenesis activity. To our knowledge, it has been the first study investigating the whole family of SLIT-ROBO proteins in AML. Methods: Expression SLIT-ROBO proteins was assessed in bone marrow biopsy specimens of 79 newly diagnosed AML patients with median age 59 years [18-87]. The paraffin-embedded tissue blocks were retrieved and subjected to immunohistochemistry for SLIT ligands (SLIT1, SLIT2, SLIT3), and their receptors ROBO1, ROBO2, ROBO3, and ROBO4. The positive blasts cells were semi-quantitatively analyzed according to previously published methods (Perrone et al, 2006). For the purpose of analysis the patients were divided into "low-expressers" and "high-expressers". Concurrently, all samples were immunostained for CD34 to calculate microvessel density (MVD) as an equivalent of angiogenesis. The control group was composed of 23 BM biopsies form patients with newly diagnosed lymphoma without bone marrow involvement. Results: Expression of ROBO receptors and SLIT ligands in AML patients and in the control group. In our study higher expression of ROBO1, ROBO2, and ROBO3 was observed more often in AML patients compared to the control group (p<0.0001, p<0.001, and p=0.09, respectively, Fig 1.). In contrast, low expression of SLIT1, SLIT2, and SLIT3 ligands has been shown more often in AML than in control BM samples (p<0.0001, p=0.003, and p=0.001, respectively,Fig.2.). Higher expression of ROBO1, ROBO2, and ROBO3 was more often in AML patients ≥60 years (p=0.04, p=0.008, and p=0.02, respectively).Conversely, low expression of ROBO4 was more often observed in elderly AML (p=0.06). The majority of patients with de novo AML had low expression of SLIT1 and SLIT2 (p=0.053 and p=0.055, respectively). As to ROBO, higher expression of ROBO2 in the group with secondary AML was more frequent (p=0.09). No significant correlations between the SLIT-ROBO proteins' expression,neither cytogenetic risk group nor clinical stage parameters such as WBC, hemoglobin level, proportion of leukemic blasts in BM, or LDH activity were found. Similarly, neither of the SLIT-ROBO proteins influenced the complete remission rate (CR) and overall survival (OS). Relationship between SLIT-ROBO expression and angiogenesis activity in AML patients and control group. Significantly higher MVD in BM of AML patients than in control group (Me 51 [9-140] vs 16 [4-78], p<0.0001) has been observed. ROBO4was the only protein that expression correlated significantly with MVD. Higher expression of ROBO4 was associated with higher MVD in both, AML and the control group (p=0.05 and p=0.01, respectively). Conclusions: SLIT-ROBO family members play a role in biology of AML. ROBO4 is involved in both, physiologic and pathologic angiogenesis A better understanding of SLIT-ROBO signaling pathway in leukemic blasts may create new optionsfor AML therapy. Acknowledgments: AG and DJ-K both equally contributed to the study. This work was supported by grants from Medical University of Lodz, Lodz, Poland (502-03/1-093-01/502-14-077 and 503/1-093-01/503-11-001). Disclosures Robak: Eisai Inc: Research Funding. Wierzbowska:Janssen, Celgene: Consultancy.

Down-Regulation of Microrna 223 and 148a Mediates Resistance of Human Acute Myeloid Leukemia Cells to Pan-Histone Deacetylase (HDAC) Inhibitors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2401-2401
Author(s):  
Jianguang Chen ◽  
Veena Kandaswamy ◽  
Warren Fiskus ◽  
Yongchao Wang ◽  
Rekha Rao ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Histone Deacetylase ◽  
Myeloid Leukemia ◽  
Hdac Inhibitors ◽  
Ectopic Expression ◽  
K562 Cells ◽  
Methyl Transferase ◽  
Human Acute Myeloid Leukemia ◽  
Acute Myeloid

Abstract Abstract 2401 Poster Board II-378 MicroRNA (miR) alterations are highly involved in the pathogenesis of leukemia. However, the role of miRs in de novo or acquired resistance of cancer cells to therapeutic agents has not been fully elucidated. Recently, we reported the isolation and characterization of HL-60/LR cells, derived from human acute myeloid leukemia HL-60 cells, that are resistant to pan-histone deacetylase (HDAC) inhibitors (HDIs), including vorinostat and panobinostat (Blood. 2008; 112: 2896). To explore the role of miRs in acquiring resistance to HDIs, we performed a miR microarray analysis of the parental HL-60 and HL-60/LR cells. Compared to HL-60 cells, expression of thirteen microRNAs were discovered to be significantly increased (> 4-fold) and fourteen miRs were markedly down-regulated (> 4-fold) in HL-60/LR cells. Alterations in the expression of three of the most promising upregulated (miR-21, miR-126 and miR-146a) and down-regulated (miR-223, miR-148a and miR-342) miRs were confirmed by Q-PCR in HL-60/LR cells. The expression of miR-223, miR-148a and miR-342 was also significant lower in the relatively HDI-resistant K562 cells as compared to HDI-sensitive U937 and HL-60 cells. Conversely, miR-126 and miR-146a expressions were higher in K562 cells compared to U937 and HL-60 cells. Short term (24 hours) treatment with panobinostat (10 to 50 nM) did not alter the expression of miR-223 or miR-148a expression in HL-60 cells. As compared to treatment with either agent alone, co-treatment with the histone methyl transferase EZH2 antagonist 3-deazaneplanocin (DZnep, 1.0 uM) and DNA methyl transferase inhibitor decitabine (2.0 uM) induced miR-223 and miR-148a levels and mediated apoptosis of HL-60/LR cells, suggesting that an epigenetic silencing mechanism(s) may be involved in the down-modulation of miR-223 and miR-148a in HL-60/LR cells. To determine whether the alterations in the miR levels were mechanistically involved in conferring resistance to HDIs, we engineered through retroviral transduction stable ectopic expressions of miR-223, miR-148a and miR-342 into HL-60/LR cells and miR-21 and miR-146a into HL-60 cells. Ectopic expression of miR-223 and miR-148a significantly increased the sensitivity of HL-60/LR cells to panobinostat and vorinostat. In contrast, re-expression of miR-342 had an insignificant effect on HDI sensitivity. Increased expression of miR-21 and miR-146a did not confer resistance to the HDIs in HL-60 and U937 cells. Next, by Western analyses, we compared the expression levels of several of the predicted target proteins of miR-223 and miR-148a, (as predicted by the computer programs TargetScan and picTAR), in HL-60 versus HL-60/LR cells, as well as in the HL-60/LR cells with stable ectopic expression of miR-223 and miR-148a. Several candidate proteins including GRP94, Ribosomal protein S6 kinase MSK1, MEF2C and DNMT1 showed higher level of expression in HL-60/LR versus HL-60 and were down-regulated in miR-223 or miR-148a transduced HL-60/LR cells, suggesting that these proteins may confer resistance against HDI. Parenthetically, miR-223 was shown to be a myeloid-specific miR which negatively regulates progenitor proliferation and granulocyte differentiation and activation. miR-223 mutant mice have an expanded granulocytic compartment resulting from an increase in the number of granulocyte progenitors. In summary, our observations indicate that high miR-223 and miR-148a levels may be predictive biomarkers for sensitivity to HDIs in human AML cells. Additionally, induction of miR-223 and miR-148a by EZH2 antagonist may increase sensitivity and overcome resistance to HDIs in human AML cells. Targeting the levels and/or activity of the miR-223 and miR-148a target proteins may also be an effective strategy in enhancing the activity of HDI based combination therapy against AML. Disclosures: Atajada: Novartis: Employment. Bhalla:Merck: Honoraria; Novartis: Honoraria, Research Funding.

Inhibition of IRAK1 As a Novel Therapeutic Strategy in Acute Myeloid Leukemia and Myelodysplastic Syndrome

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 612-612 ◽  
Author(s):  
Garrett Rhyasen ◽  
Lyndsey Bolanos ◽  
Jing Fang ◽  
Carmen Rigolino ◽  
Agostino Cortelezzi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Protein Expression ◽  
Myelodysplastic Syndrome ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Activity ◽  
Myeloid Malignancies ◽  
Cd34 Cells ◽  
Acute Myeloid

Abstract Abstract 612 Recent work has shown that acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients exhibit downregulation of miR-146a, a miRNA that negatively regulates the innate immune pathway by targeting IRAK1 and TRAF6. Mice lacking miR-146a show elevated IRAK1 protein expression, and develop AML and MDS-like features resembling the human diseases. Prior to this study, the role of IRAK1 in human myeloid malignancies was unknown. We conducted a comparison of gene expression profiles of 136 cases of MDS CD34+ cells with 17 normal CD34+ cells obtained from ArrayExpress (E-GEOD-19429; Pellagatti et al., Leukemia, 2010). According to this data set, we observed IRAK1 overexpression in MDS patients (P = 0.017). IRAK1 is a serine/threonine kinase, and after phosphorylation on threonine-209 (T209), its kinase activity is induced, thus allowing for subsequent activation of TRAF6 and eventually NF-kB. Interestingly, we observed higher basal levels of phospho-IRAK1 at T209 in MDS and AML samples as compared to normal human CD34+ cells. To investigate the potential role of IRAK1 in AML and MDS, we used genetic and pharmacological approaches to suppress IRAK1 activity in MDS/AML cell lines and bone marrow cells from MDS patients. RNAi-mediated knockdown of IRAK1 in MDS and AML samples resulted in impaired growth of malignant hematopoietic stem/progenitor cells in methylcellulose assays and rapid apoptosis in vitro. In addition, we used a small-molecule inhibitor (benzimidazole analog; Amgen Inc.) to potently inhibit IRAK1 kinase activity. MDS/AML cell lines and MDS patient samples cultured with the IRAK1 inhibitor exhibited impaired growth and increased apoptosis, which coincided with decreased phospho-IRAK1 at T209, and active versions of TRAF6 and NF-kB. Importantly, the inhibition of IRAK1 kinase function is selectively detrimental to MDS and AML samples while preserving normal CD34+ cell viability and function. Given this novel requirement of IRAK1 in MDS and AML, we examined whether Lenalidomide or Bortezomib, two treatment options for MDS/AML and reported immunosuppressors, exhibit anti-leukemic activity in part by targeting IRAK1. We observed that Bortezomib, but not Lenalidomide, inhibits IRAK1 mRNA and protein expression in MDS/AML cells. The cytotoxic effect of Bortezomib can be partly rescued by forced expression of IRAK1 in these cells. To determine the molecular and cellular basis of cell death following loss of IRAK1 function or expression, we applied microarrays to MDS cells treated with IRAK1 inhibitor or transduced with a lentiviral vector encoding an shRNA targeting IRAK1. An overlap of commonly deregulated genes imposed by loss of IRAK1 expression or by the IRAK1 inhibitor revealed unique pathways relevant to the survival of MDS and AML cells. In summary, these findings are the first to implicate IRAK1 in the maintenance of myeloid malignancies and describe the effectiveness of an IRAK1 inhibitor on suppressing MDS and AML viability. Disclosures: Oliva: Celgene: Consultancy.

scholarly journals The Role of IDH Mutants, Which Are Promising Therapeutic Targets for Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3796-3796
Author(s):  
Yoko Ogawara ◽  
Hironori Matsunaga ◽  
Takahiko Seki ◽  
Yukino Machida ◽  
Kazushi Araki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Mouse Model ◽  
Myeloid Leukemia ◽  
Myeloproliferative Neoplasms ◽  
Idh Mutations ◽  
Mutant Idh1 ◽  
Mutant Genes ◽  
Acute Myeloid

Abstract Mutations in isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in acute myeloid leukemia (AML), glioma, and many other cancers. While wild-type IDHs convert isocitrate to α-ketoglutarate (α-KG), mutant IDHs convert α-KG to oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases, such as TETs, histone demethylases, EGLNs, and other enzymes. Because the role of mutant IDH is not necessary for normal cells, inhibitors directed against mutant IDH are not expected to have the side effects as those of anti-cancer agents. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of mutant IDH-dependent AML. Previously, the IDH mutation alone was shown to be insufficient for the induction of AML, and IDH mutations occur simultaneously with mutations in other genes such as NPM, DNMT3A, and FLT3. In accordance with these observations, we found that NPM+/- hematopoietic progenitor cells transduced with IDH2/R140Q, NPMc, DNMT3A/R882H, and FLT3/ITD cooperatively induced AML in a mouse model. However, when only three of these mutant genes were transduced, myeloproliferative neoplasms (MPNs) rather than AML was more frequently induced and their onset was delayed in any combinations of the mutant genes. These results clearly indicate that all four mutations are necessary for the efficient induction of AML. By using a combination of AML model mice with cre-loxp, we conditionally deleted IDH2/R140Q from AML mice, which blocked 2-HG production and resulted in the loss of leukemia stem cells. Accordingly, the progression of AML was significantly delayed. These results indicate that the function of IDH2 mutation is critical for the development and maintenance of AML stem cells, and that mutant IDHs are promising targets for anticancer therapy. Based on these findings, we developed potent and specific inhibitors of mutant IDH1 and tested their effects in the mutant IDH1-dependent AML mouse model, created by introducing four mutant genes including mutant IDH1. The 2HG level was promptly and dramatically decreased in AML cells soon after treatment with the mutant IDH1 inhibitors, and the number of leukemia cells was reduced after a 4-week treatment. These results indicate that IDH1 mutant inhibitors are effective for the treatment for AML. Because IDH mutations and TET2 mutations are mutually exclusive in AML, the inhibition of TET-mediated conversion of 5mC to 5hmC is considered one of the main roles of mutant IDH. We found that levels of 5hmC on differentiation-inducing genes, such as Ebf1, Spib and Pax5 were decreased in AML cells with IDH2/R140Q and recovered by conditional deletion of IDH2/R140Q. In consistent with levels of 5hmC, expressions of these genes are downregulated in the AML cells and increased by deletion of IDH2/R140Q. Gene expression analysis revealed that IDH2/R140Q up-regulates a set of genes that is activated in response to hypoxia as well as Meis1. As 2HG inhibits EGLN that hydroxylates and marks HIF1α for ubiquitin-proteasomal degradation, it is probable that mutant IDH2-produced 2HG stabilizes HIF1α through inhibition of EGLN. Furthermore, it was reported that Meis1 activates the transcription of HIF1α. In consistent with these information, we showed that IDH2/R140Q increased the protein levels of HIF1α in cultured cells. Disclosures Matsunaga: Daiichi Sankyo Co., Ltd.: Employment. Seki:Daiichi Sankyo Co., Ltd.: Employment. Araki:Daiichi Sankyo Co., Ltd.: Employment. Kitabayashi:Daiichi Sankyo Co., Ltd.: Research Funding.

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