CD99 Is Highly Expressed in Acute Myeloid Leukemia (AML) and Presents a Viable Therapeutic Target

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1540-1540 ◽  
Author(s):  
Vijaya Pooja Vaikari ◽  
Miran Jang ◽  
Mojtaba Akhtari ◽  
Houda Alachkar
Keyword(s):  
Bone Marrow ◽  
Therapeutic Target ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Normal Bone Marrow ◽  
Potential Therapeutic Target ◽  
Normal Bone ◽  
Healthy Donors ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by clonal proliferation and block of differentiation of myeloid precursors. Overall survival for patients with AML remains dismal (<50% for younger patients and <10% for older patients) due to high relapse rate. In search for novel therapeutic targets in AML, we compared gene expression data of normal hematopoietic vs AML cells from 7 datasets (GSE13159, GSE13164, GSE7186, GSE1159, GSE995, GSE31174 and TCGA Leukemia) available on Oncomine. We identified CD99 to be significantly upregulated in AML cells compared with normal cells in all data sets with available measurements of CD99 expression (median ranking 155, p = 0.013); other genes among the top 10 genes identified in this analysis were FLT3 (median ranking 102, p<0.001) and WT1 (median ranking 120, p<0.001); both are known to play a role in AML. CD99 was significantly over-expressed (p<0.001) in 542 AML patients as compared with PBMCs from 74 healthy donors from the GSE13159 dataset. In the GSE13164 dataset, CD99 was significantly over-expressed in 257 AML patients as compared with PBMCs from 58 healthy donors. Consistently, in the GSE7186 dataset, CD99 was significantly over-expressed (p<0.001) in 23 AML patients as compared with 6 normal bone marrow samples and in the GSE1159 dataset, CD99 was significantly over-expressed (p=0.001) in 285 AML patients as compared with 5 normal bone marrow, and 3 normal blood samples. We also analyzed CD99 expression in cells obtained from 23 patients with AML and sorted according to their CD34 and CD38 expression levels (GSE3077 dataset). We found that CD99 expression was significantly higher (p<0.001) in the CD34+CD38+ and CD34+CD38- subpopulation compared with CD34-CD38- and CD34-CD38+; suggesting a possible role of CD99 in AML stem cells. Interestingly, analysis of three datasets (GSE22848, GSE6891, GSE15434) via R2: Genomics Analysis and Visualization Platform showed a correlation between CD99 expression and the presence of FLT3-ITD mutation. In the GSE22848 dataset, CD99 was significantly over-expressed (p=0.007) in 48 patients with FLT3-ITD as compared with 189 patients with FLT3 wildtype. In the GSE6891 dataset, 126 patients with FLT3-ITD had a significant over-expression of CD99 (p=0.006) as compared with 334 patients with FLT3 wildtype and in the GSE15434 dataset 90 FLT3-ITD positive patients had significantly higher levels of CD99 (p<0.001) as compared with 161 patients with the wildtype gene. CD99 (E2, MIC2), a 32-kD cell surface glycoprotein, is known to be involved in the transendothelium migration of neutrophils, T-cell adhesion, and T-cell death by a caspase-independent pathway. In cancer cells, CD99 was found to be highly expressed on the cell surface of Ewing's sarcoma tumors and in gliomas. Importantly, CD99 expression levels were found to be correlated with tumor invasiveness and with lower survival rates. In order to examine the role of CD99 in AML, we assessed CD99 expression by flow cytometry in nine AML cell lines (KG-1, KG-1A, MOLM13, MV4-11, Kasumi-1, THP-1, NB4, U937, UOC-M1); we found CD99 to be expressed in all cell lines. To determine whether CD99 is a potential therapeutic target in AML, we treated leukemia cells with anti-CD99 mAb (mAb 0662) at 5 µg/mL and analyzed cell viability 48 hours post-treatment. We found significant decrease in cell viability; 15% in MV4-11 cells (p=0.02), 32% in MOLM13 cells (p=0.002) and 18% in THP-1 cells (p<0.001) as compared with untreated controls measured by Alamar blue assay. Furthermore, inhibiting CD99 led to a decrease in migration of MV4-11, MOLM13 and THP-1 cells when analyzed using a trans-well migration assay. In conclusion, CD99 is highly expressed in AML, and this expression is significantly higher in less differentiated leukemia cells and in patients with FLT3-ITD mutation. Functional studies using CD99 antibodies revealed a possible role of this gene in cell survival and cell migration. Further studies are needed to establish CD99 as a potential therapeutic target and further investigations are ongoing to determine the mechanism by which CD99 regulates cell survival in AML. Disclosures No relevant conflicts of interest to declare.

VEGF-A, VEGF-C, and CD31 Expression by Leukemia Cells Are Prognostic Factors for Overall Survival in Acute Myeloid Leukemia (AML).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2392-2392
Author(s):  
Eunice S. Wang ◽  
Amanda Sobczyk ◽  
Joseph M. Marinaro ◽  
Meir Wetzler ◽  
Lili Tian ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Prognostic Factors ◽  
Myeloid Leukemia ◽  
Fold Increase ◽  
Leukemia Cells ◽  
Normal Bone Marrow ◽  
Bone Marrow Biopsies ◽  
Normal Bone ◽  
Acute Myeloid

Abstract INTRODUCTION: Bone marrow angiogenesis has increasingly been recognized as important for leukemogenesis. Identification of vascular endothelial growth factor (VEGF) mediated autocrine and paracrine loops in subsets of acute myeloid leukemia (AML) support multiple mechanisms of action for these factors. We examined whether expression of pro-angiogenic growth factors VEGF-A, VEGF-C, and angiopoietin-2 (Ang-2) correlated with marrow neovascularization (CD31 staining) and overall survival of AML patients (pts). PATIENTS AND METHODS: Expression of VEGF-A, VEGF-C, and Ang-2 was determined in leukemia bone marrow samples at diagnosis by quantitative real-time reverse transcription polymerase chain reaction (Q-PCR) using 18s RNA controls. Expression was then analyzed for fold-differences versus normal bone marrow control samples (n=10). VEGF-A and VEGF-C protein expression in leukemia cells and CD31 protein expression in microvessels and leukemia cells was evaluated by immunohistochemistry (IHC) in the corresponding bone marrow biopsies from the same pts. A cohort of 96 AML pts (46 male, 50 female) was examined with a median age of 67.5 years (range 21–96 years). Half (n=46) achieved an initial complete remission (CR) on standard induction chemotherapy regimens. Median overall survival was 9.3 months. No pts had recurrent cytogenetic abnormalities, and 76 had normal karyotype. RESULTS: Leukemia blast VEGF-A (median 1.77 fold increase, range 0.06–23.62) and Ang-2 (median 6.9 fold increase, range 0.07–3100) expression by Q-PCR were consistently higher than normal bone marrow controls in contrast to VEGF-C that was lower than normal controls (median 0.51, range 0.04–27.93). Using a cut-off of 1.5 fold to denote growth factor overexpression by Q-PCR, we found that prolonged pt survival correlated with low VEGF-C levels (mean 609 ± 92 n=78 vs. high VEGF-C 235 ± 46 days, n=18, p =0.056) and high Ang-2 levels (mean 581± 91, n=77 vs. low Ang-2 366 ± 98 days, n=19 vs. p=0.26) but was not statistically significant. No difference in survival was noted in low vs. high VEGF-A expressing samples (mean 545 + 171, n=26, vs. 511 + 105, n=70, p=0.96). No correlation between CD31+ microvessels and survival was noted. By IHC, most leukemic marrow biopsies were positive for VEGF-A (n=60) and negative for VEGF-C (n=87) and CD31 (n=78). Univariate analysis of overall survival using Cox proportional hazard model demonstrated that age (p=0.0008), achievement of CR (p&lt;0.0001), karyotype (normal vs. complex/other, p=0.0210), and VEGF-A (p=0.0272), VEGF-C (p=0.0255), and CD31 positive IHC expression by AML blasts (p=0.0152) were independent prognostic factors. Multivariate analysis showed that the only factors of statistical significance were achievement of CR (hazard ratio 4.09, 95% CI 2.28–7.33, p &lt;0.0001) and VEGF-C expression by IHC (hazard ratio 0.08, 95% CI 0.01-0.81). CONCLUSIONS: These results show that expression of VEGF-A, VEGF-C, and CD31 (in AML blasts) by IHC in bone marrow biopsies represent prognostic factors for overall survival in AML. The importance of VEGF-C mediated angiogenic processes as an independent prognostic factor in AML outcome suggests that targeted inhibition of this factor may be of future therapeutic benefit in subsets of AML pts.

The Role of TWIST1 Gene Expression and Hypoxic Microenvironment in Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3839-3839
Author(s):  
Emilia Carolina Malafaia ◽  
A. Mario Marcondes ◽  
Ekapun Karoopongse ◽  
Daniele Serehi ◽  
Maria de Lourdes L. F. Chauffaille ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Twist1 Expression ◽  
Disease Biology ◽  
Acute Myeloid

Abstract TWIST1, a basic helix-loop-helix (bHLH) transcription factor, plays a critical role in mesodermal development and organogenesis. Overexpressed TWIST1 has been thoroughly related to epithelial-mesenchymal transition (EMT) in solid tumors (QIN Q et al., 2012) and has been described as an emerging risk factor in hematological neoplasms (MERINDOL et al., 2014). . Many questions remain to be addressed concerning to the role of TWIST1 in acute myeloid leukemia (AML). The understanding of TWIST1 in leukemia cells and its interaction with microenvironment can offer new insights in regards to disease biology and therapeutic targets for patients with AML. Objectives: 1) to evaluate the role of stroma contact and hypoxia in TWIST1 expression in myeloid cell lines. 2) To evaluate the functional impact of overexpressing TWIST1 on KG1a and PL21 cells. 3) To evaluate TWIST1 expression in primary cells of AML patients. Methods: In order to mimic bone marrow microenvironment, myeloid cells were co-cultured with mesenchymal HS5 cell line and PO2 1% was established with Smart -Trak¨ 2 (Sierra Instruments, Inc.) equipment. Quantitative mRNA was determined using TaqMan¨ Universal Master Mix (Applied Biosystems, Foster City, CA) and 3-step standard cycling conditions with sequence-specific primer TWIST1 normalized to the expression of β-actin. KG1a and PL21 cells were transduced with lentivirus vector carrying e-GFP ("enhanced green fluorescence protein") for stable expression of TWIST1. Transduced cells were sorted by FITC fluorochrome and then verified through western blot analysis with TWIST1 antibody. For quantification of apoptosis, cells were labeled with PE-conjugated antibody using annexin V-phycoerythrin and propidium iodide (BD Biosciences, USA). DAPI (4',6- diamidino-2-phenylindole dihydrochloride) was used to stain DNA and determine cell cycle information . Apoptosis and cell cycle were analyzed by FACS -Becton Dickinson Canto II (BD Biosciences). Statistical analysis was assessed with unpaired t test. Results: Hypoxia induced TWIST1 mRNA expression in OCIAML3, PL21, KG1a and ML1 cell lines (fold-increased 46.3, 29.8, 12.9 and 2.3 respectively). Cells expressing endogenous TWIST1 protein (OCIAML3 and ML1) showed resistance to apoptosis in a hypoxic microenvironment (normoxia versus hypoxia: OCI/AML3, 22.6 % vs 11.7% and ML1, 29.8% vs. 7.5%) in contrast, cells not expressing endogenous TWIST1 protein (KG1a and PL21) went to apoptosis in the same conditions. Thus, overexpressing TWIST1 in KG1a and PL21 induced apoptosis protection in hypoxia (KG1a unmodified vs. modified: 17.6 ± 6.3 vs. 2.8 ± 6.3, p=0.04; PL21 unmodified vs. modified: 26.9 ± 10.9 vs. 3.2 ± 0.6, p=0.04) (fig 1). We found increased TWIST1 mRNA levels in bone marrow samples of 23 AML patients (3.88 ± 1.59) compared with 5 healthy controls (0.54 ±0.25) (p= 0.02) (fig 2). Patients in the highest tertile of TWIST1 expression did not show differences in percentage of blasts in bone marrow and complete remission after treatment compared with patients in low and middle tertile. Conclusion: Our data suggest TWIST1 gene expression protects acute myeloid leukemia cells from apoptosis in a hypoxic microenvironment. Moreover, our results showed increased expression of TWIST1 in AML patients. Thus, TWIST1 is a potential gene involved in leukemogenesis and should be further explored to understand disease biology and potential therapeutic targets. Disclosures No relevant conflicts of interest to declare.

scholarly journals Identification of CHD4 As a Potential Therapeutic Target of Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1648-1648 ◽  
Author(s):  
Yaser Heshmati ◽  
Gözde Turköz ◽  
Aditya Harisankar ◽  
Sten Linnarsson ◽  
Marios Dimitriou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Loss Of Function ◽  
Potential Therapeutic Target ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is characterized by impaired myeloid differentiation of hematopoietic progenitors, causing uncontrolled proliferation and accumulation of immature myeloid cells in the bone marrow. Rearrangements of the mixed lineage leukemia (MLL) gene are common aberrations in acute leukemia and occur in over 70% in childhood leukemia and 5-10% in leukemia of adults. MLL rearrangements encode a fusion oncogenic H3K4 methytransferase protein, which is sufficient to transform hematopoietic cells and give rise to an aggressive subtype of AML. Leukemia where the MLL fusion oncogene is expressed is characterized by dismal prognosis and 30-60% of 5-years overall survival rate. The current standard treatment for AML is chemotherapy and in certain cases bone marrow transplantation. However, chemotherapy causes severe side effects on normal cells and an increased risk of relapse. Consequently, discovery of novel drug targets with better efficacy and low toxicity are needed to improve treatment of AML. In this study, we aimed to identify genes that are required for growth of AML cells and that encode proteins that potentially could be used as therapeutic targets. To do this, we performed high-throughput RNAi screening covering all annotated human genes and the homologous genes in mice, using barcoded lentiviral-based shRNA vectors. Stable loss-of-function screening was done in three AML cell lines (two human and one murine AML cell lines) as well as in a non-transformed hematopoietic control cell line. The candidate genes were selected based on that shRNA-mediated knockdown caused at least a 5-fold growth inhibition of leukemic cells and that the individual candidates were targeted by multiple shRNAs. The chromodomain Helicase DNA binding protein 4 (CHD4), a chromatin remodeler ATPase, displayed the most significant effect in reduced AML cell proliferation upon inhibition among the overlapping candidate genes in all three AML cell lines. CHD4 is a main subunit of the Nucleosome Remodeling Deacetylase (NuRD) complex and has been associated with epigenetic transcriptional repression. A recent study has shown that inhibition of CHD4 sensitized AML cells to genotoxic drugs by chromatin relaxation, which increases rate of double-stranded break (DSB) in leukemic cells. To verify whether CHD4 is exclusively essential for AML with MLL rearrangements, we inhibited CHD4 expression with two independent shRNAs in various AML cell lines with and without MLL translocations. In vitro monitoring of growth and viability indicated that knockdown of CHD4 efficiently suppressed growth in all tested cell lines, suggesting that CHD4 is required in general for growth of leukemic cells. To test the effect of CHD4 inhibition in normal hematopoiesis, we pursued knockdown of CHD4 and monitored effects in hematopoiesis using colony formation assays of human CD34+ cells. The results demonstrated that CHD4 knockdown had minor effects in colony formation as well as growth and survival of normal hematopoietic cells. Furthermore, to explore whether inhibition of CHD4 can prevent AML tumor growth and disease progression in vivo, we have generated a mouse model for AML. By transplanting AML cells transduced with shRNA against CHD4 into recipient mice, we showed that shRNA-mediated targeting of CHD4 not only significantly prolonged survival of AML transplanted mice but also in some cases completely rescued some mice from development of the disease. Collectively, these data suggested that CHD4 is required for AML maintenance in vivo. Next, to determine whether suppression of CHD4 can inhibit cell growth of different subpopulations and subtypes of AML, we performed loss of function studies of CHD4 on patient-derived AML cells ex vivo. Loss of CHD4 expression significantly decreased the frequency of leukemic initiating cells in different subtypes AML patient samples. In further in vivo studies using a xeno-tranplantation model for AML, we demonstrated that shRNA-mediated inhibition of CHD4 significantly reduced the frequency of leukemic cells in the marrow 6 weeks after transplantation. Taken together our results demonstrated the critical and selective role of CHD4 in propagation of patient-derived AML cells as well as in disease progression in mouse models for AML. We believe that CHD4 represents a novel potential therapeutic target that can be used to battle AML. Disclosures No relevant conflicts of interest to declare.

Knockdown of Stromal Interaction Molecule 1 (STIM1) Suppresses Acute Myeloid Leukemia Cell Line Survival Through Inhibition of Reactive Oxygen Species Activities

2021 ◽  
Author(s):  
Eman Salem Algariri ◽  
Rabiatul Basria S. M. N. Mydin ◽  
Emmanuel Jairaj Moses ◽  
Simon Imakwu Okekpa ◽  
Nur Arzuar Abdul Rahim ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Acute Myeloid Leukemia ◽  
Cell Line ◽  
Therapeutic Target ◽  
Myeloid Leukemia ◽  
Oxygen Species ◽  
Down Regulation ◽  
Potential Therapeutic Target ◽  
Acute Myeloid

Abstract Stromal interaction molecule 1 (STIM1) is a critical regulator of calcium homeostasis through store-operated calcium entry (SOCE) and recently considered a potential therapeutic target for cancer. However, the role of STIM1 in acute myeloid leukemia (AML) remains unclear. The present study investigates the role of STIM1 in AML cell line (THP-1) proliferation and survival and its effect on reactive oxygen species (ROS) activities. Dicer-substrate siRNA (dsiRNA) - mediated STIM1 knockdown inhibited the THP-1 cells proliferation and colony formation ability. Further observation on ROS profile showed a significant reduction in the ROS level, which was associated with a significant down-regulation of NOX2 and protein kinase C (PKC). Furthermore, STIM1 knockdown exhibited significant down-regulation of Akt, KRAS, and MAPK which are critical proliferative and survival pathway-related genes. This study unveiled the importance of STIM1 in the regulation of AML cells proliferation and survival which could be through maintaining ROS at level keeping the proliferative and survival pathways at an active state. These findings represent STIM1 as a potential therapeutic target for AML treatment.

PU.1 and C/EBPalpha Expression in Acute Myeloid Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4509-4509
Author(s):  
Annalisa Di Ruscio ◽  
Francesco D’Alò ◽  
Francesco Guidi ◽  
Emiliano Fabiani ◽  
Giuseppe Leone ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Transcription Factors ◽  
Myeloid Leukemia ◽  
Normal Karyotype ◽  
The Other ◽  
Mrna Levels ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Acute Myeloid

Abstract The myeloid transcription factors C/EBPalpha and PU.1 play a pivotal role in normal hematopoiesis and alterations of their function are involved in the pathogenesis of Acute Myeloid Leukemia (AML). So far, different mechanisms have been shown to affect their function and are important in some AML subsets. However most AML patients do not apparently show any alteration of these transcription factors. Here, we investigated C/EBPalpha and PU.1 mRNA levels by real time RT-PCR in 109 AML patients and correlated these data to morphology, FLT3 mutations and cytogenetics. C/EBPalpha and PU.1 levels were expressed as percentage of 18S. Twelve normal bone marrow mononuclear cells, four CD34+ cells isolated from normal bone marrow samples and 8 peripheral blood granulocytes and monocytes, were used as controls. Heterogeneous PU.1 expression was observed in AML patients (median 0.657, range 0.004 – 24.148), while PU.1 levels were more homogeneous in normal bone marrows (median 1.5, range 0.328 – 4.737). In particular, 55 AML patients (50.5%) had PU.1 levels similar to controls, while 37 patients (33.9%) and 17 patients (15.6%) expressed PU.1 levels at levels lower and higher, than the control range, respectively. In the same way, also C/EBPalpha mRNA expression was variable (median 0.047, range 0.0002 – 1.858 in AML and median 0.064, range 0.008 – 0.138 in normal bone marrows). Fourty-five AML patients (41.%) displayed C/EBPalpha levels similar to the normal range, while 26 patients (23.8%) had lower and 37 (33.9%) higher C/EBPalpha expression. Looking at different AML subsets, we found low C/EBPalpha mRNA in patients carrying recurrent chromosomal abnormalities, such as t(8;21) and inv16, as previously reported. On the other hand, patients carrying 11q23 rearrangements showed higher PU.1 levels than normal controls. No association was found between C/EBPalpha and PU.1 levels and therapy-related AML, AML with normal karyotype, AML with multilineage dysplasia, and AML not otherwise characterized (including previous F.A.B. categories). Although experimental models showed that FLT3 internal tandem duplications (ITD) downregulate both transcription factors, we did not find any association between the presence of FLT3 ITD and D835 mutations and C/EBPalpha and PU.1 levels, both in the whole patient group and in patients with normal karyotype. We then analyzed expression of two PU.1 and C/EBPalpha target genes, the M-CSF and G-CSF receptors, in patients expressing high and low levels of these transcription factors. A direct correlation was found between C/EBPalpha and G-CSFR levels (Spearman r = 0.5; p=0.02, 95% C.I. 0.07 – 0.78), while there was a tendency to correlation between PU.1 and M-CSFR, that did not reach the statistical significance. Since mutations and post-trascriptional events may affect C/EBPalpha and PU.1 function, we analyzed protein expression of 18 patients by Western Blotting. PU.1 protein was expressed by all patients. The functional p42 C/EBPalpha isoform was absent in 2 patients that expressed only the 30 kDa isoform, and was undetectable in 5 of 18 patients. In conclusion, down regulation of PU1 mRNA was found in one third of AML patients, consistently with the oncosuppressive role recently described. On the other side, C/EBPalpha is down-regulated in specific AML subsets, with recurrent cytogenetic abnormalities, while mutations and post-translational events could affect C/EBPalpha expression in other patients.

Transglutaminase2 Expression in Acute Myeloid Leukemia: Association with Adhesion Molecule Expression and Leukemic Blast Motility

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1427-1427
Author(s):  
Steven M. Kornblau ◽  
Andrew Pierce ◽  
Stefan Meyer ◽  
Farhad Ravandi ◽  
Gautam Borthakur ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Normal Bone Marrow ◽  
Adhesion Proteins ◽  
Entire Cohort ◽  
Normal Bone ◽  
Cd34 Cells ◽  
Acute Myeloid

Abstract Abstract 1427 Background: In prior proteomic analysis on Acute myeloid leukemia (AML) cell lines evaluating the effects of several leukemogenic oncogenes we observed that transglutaminase2 (TG2) was expressed at greater levels as a consequence of oncogenic transformation. TG2 is a multi-domain, multi-functional enzyme with diverse biological functions, including extracellular matrix formation, integrin-mediated signalling, and signal transduction. It's normal roles remain obscure, but it is linked to the pathogenesis of celiac sprue, neurodegenerative diseases, and some cancers. In malignancy it is reported to be an anti-apoptotic mediator of hypoxia inducible factor (HIF) conferring a growth advantage to tumor cells. Expression has been associated with resistance to chemotherapy and apoptosis. We therefore assess the expression of TG2 protein in primary AML patient samples. METHODS: We analyzed 511 AML samples from patients with newly diagnosed AML using a custom made reverse phase proteomic array. This array included 11 normal bone marrow derived CD34+ samples as controls and had 140 paired same day blood and marrow samples and 49 paired diagnosis and relapse samples. The array was probed with antibodies against 203 targets including TG2 (Abcam, ab2386, UK). Supercurve algorithms were used to generate a single value from the five serial dilutions. Loading control and topographical normalization procedures accounted for protein concentration and background staining variations. RESULTS: Expression of TG2 was statistically similar (p= 0.43) in paired blood and marrow samples and in protein prepared from fresh cells or from cryopreserved cells (p= 0.71). Expression was above, equal to or below that of normal CD34+ cells in 12%, 62%, and 27% of patients. Levels were significantly higher at relapse compared to diagnosis in the 49 paired samples (p = 0.003). Levels were higher in FAB M6 and M7 (P =<0.00001 and < 0.008) and lower in patients with inversion16. Higher TG2 expression was strongly inversely correlated with total WBC (r=.035, p < 0.0001) and the absolute blood blast count (r = −.30, p <0.0001). Patients with higher TG2 level had a shorter but not statistically significant overall survival in the entire cohort, and was not prognostic in subsets stratified based on cytogenetics or mutations (FLT3, NPM1, RAS). Likewise, patients with higher TG2 levels had shorter remission durations, but again this was not significant in the entire cohort or in subsets. Expression of TG2 was significantly correlated with 55 of 203 proteins. Notable among these were numerous integrin and adhesion proteins. Hierarchical clustering of these demonstrated that AML is characterized by two large cohorts, one in which TG2 is elevated and is positively correlated with CD49B, Integrinβ3, FAK, Fibronectin and IGFB2, a second in which TG2 is low and negatively correlated with high expression of Osteopontin, CD11 and, CD44 and a 3rd in which only Caveolin1 is expressed. A Cytoscape interaction plot based on online databases of known protein-protein interactions revealed that TG2 has known interactions with Fibronectin, which it binds and post-translationally modifies, and integrinβ3. In combination this suggests that there is canonical interaction between TG2 and integrin and adhesion proteins active in AML. TG2 expression also correlated positively with numerous anti-apotptosis proteins. CONCLUSION: TG2 is expressed in the majority of cases of AML at levels comparable to normal bone marrow CD34+ cells and levels became significantly higher at relapse suggesting that the protein expression signature associated with high TG2 levels may be selected for, or confer a subtle survival advantage to leukemic blasts. In support of this, while the level of TG2 was not statistically significantly prognostic for either overall survival or remission duration, patents with higher levels were somewhat more likely to relapse, and less likely to be alive beyond 3 years. TG2 has previously been linked to drug resistance in cancer and given the negative correlation between TG2 levels and peripheral blasts observed increased TG2 levels may lead to the protection of the leukemic stem cell due to increased adhesion/reduced motility. TG2 may therefore form part of a network of proteins that define poor outcome in AML patients and potentially offer a target to sensitize AML stem cells to drug treatment. Disclosures: Off Label Use: Clofarabine in AML.

Interleukin-4 Is a Negative Regulator of Acute Myeloid Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2146-2146
Author(s):  
Pablo Enrique Peña Martínez ◽  
Marion Chapellier ◽  
Mia Eriksson ◽  
Ramprasad Ramakrishnan ◽  
Carl Högberg ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Negative Regulator ◽  
Interleukin 4 ◽  
Leukemia Cells ◽  
P Value ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Marrow Cells

Abstract Acute myeloid leukemia (AML) is the most common acute leukemia among adults and is associated with poor prognosis. Hence, there is a strong need to develop new therapeutic strategies towards improved treatments. In this study, we performed an in vitro cytokine screen designed to identify selective negative regulators of leukemia cells. By mixing murine AML cells driven by the MLL-AF9 fusion gene in a dsRed transgenic background and non-dsRed normal bone marrow cells, we screened 114 murine recombinant proteins in a 96-well format and assessed their effect on cell expansion during 72 hours. Both the leukemia cells and the normal bone marrow cells were enriched for primitive cells by isolating C-kit positive cells. In the screen, Interleukin 4 (IL-4) emerged as the top hit, as it caused the strongest depletion of leukemia cells while maintaining the normal bone marrow cells. Further validations demonstrated that the negative effect induced by IL-4 on leukemia proliferation was obtained even in the presence of cytokines such as IL-3, which alone strongly stimulates leukemia cell growth and survival. In contrast to the negative effects of IL-4 on leukemia cells, IL-4 alone was validated to be sufficient to maintain cell survival of normal bone marrow cells in short term cultures. To assess the mechanisms by which IL-4 depletes leukemia cells, we carried out an apoptosis analysis upon stimulating the leukemia cells with IL-3 or a combination of IL-3 and IL-4. The percentage of late apoptotic cells was significantly increased by IL-4, suggesting that this cytokine pushes the cells into an apoptotic state. To explore which signaling pathway is responsible for the IL-4-induced effect in leukemia cells, we performed phospho-flow analysis on Stat6, one of the main effectors downstream of the IL-4 receptor. IL-4 stimulation induced strong phosphorylation of Stat6, suggesting that this pathway may be responsible for the IL-4-induced effects in leukemic cells. To address whether IL-4 stimulation of leukemia cells affects leukemia initiating cells, also termed leukemia stem cells, we next stimulated leukemia cells ex vivo for 3 days with IL-4, IL-3, or no cytokines, followed by transplantation of the cells to sublethally irradiated mice (n= 6 in all groups). Mice receiving IL-4 treated cells had a significant prolonged survival (p-value= 0.0003, median= 37 days) than those receiving cells treated with IL-3 or non-treated cells (median= 27 and 30 days, respectively). We next performed an in vivo treatment experiment with intra-peritoneal injections of IL-4 at a dose of 60 μg/Kg/day. Mice were treated daily for 10 days and sacrificed at day 13. Mice treated with IL-4 (n= 6) had lower percentage of MLL-AF9 cells both in blood (36±4.5%) and bone marrow (89±2,8%) compared to control mice (53±4.1% in blood, 96±0.6% in bone marrow, n= 6). We then treated mice with escalating doses of IL-4 (15, 30, and 60 μg/Kg/day, n= 5) and PBS as control (n= 7) and monitored their survival. Only the group receiving 60 μg/Kg/day had a significant prolonged survival (p-value= 0.0077, median= 26 days) when compared to the control group (median= 23 days). In summary, these findings demonstrate that IL-4 is a negative regulator of AML cells in this murine MLL-AF9 leukemia model. Future experiments will explore the mechanisms underlying these effects and its relevance for human disease. Disclosures No relevant conflicts of interest to declare.

Ocular relapse in acute myeloid leukemia (M4) with normal bone marrow

2008 ◽  
Vol 29 (4) ◽  
pp. 243-245 ◽  
Author(s):  
Hayyam Kiratli ◽  
Haluk Demiroğlu ◽  
Serkan Emeç
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Acute Myeloid
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