Differential effect of GM-CSF pretreatment on intracellular Ara-C metabolism in normal bone marrow mononuclear cells vs acute myeloid leukemia (AML) blasts

Abstract Acute myeloid leukemia (AML) is a biologically heterogeneous disease of the hematopoietic system characterized by a clonal accumulation of immature blast cells in the bone marrow. In this study, we used a proteomic approach based on two-dimensional electrophoresis and mass spectrometry to search for biomarkers related to the complete remission (CR) state of AML patients. We detected one AML-related protein spot using two-dimensional electrophoresis analysis, which is identified as the B-cell translocation gene 1 (BTC1) protein that belongs to anti-proliferative protein family. In the CR state of AML-m2 and M3 patients, BTG1 protein was upregulated in the bone marrow mononuclear cells. In addition strong BTG1 protein spot was detected in the normal bone marrow mononuclear cells while BTG1 levels in samples from AML-M2 patients in the non-remission state after therapy were not increased compared to those prior to therapy. Overexpression of BTG1 mRNA was also observed in the CR state of all-trans retinoic acid (ATRA)-treated AML-M3 patients and ATRA-treated HL-60 cells. Taken together, these results suggest that BTG1 may play a role in the differentiation process of myeloid cells and thus be a potential treatment-related biomarker for monitoring the remission status of AML-M2 and M3 patients following appropriate therapy.

Download Full-text

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

Blood ◽

10.1182/blood.v108.11.4509.4509 ◽

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.

Download Full-text

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

Blood ◽

10.1182/blood.v120.21.1427.1427 ◽

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.

Download Full-text

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

International Ophthalmology ◽

10.1007/s10792-008-9207-5 ◽

2008 ◽

Vol 29 (4) ◽

pp. 243-245 ◽

Cited By ~ 6

Author(s):

Hayyam Kiratli ◽

Haluk Demiroğlu ◽

Serkan Emeç

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Normal Bone Marrow ◽

Normal Bone ◽

Acute Myeloid

Download Full-text

Vitamin D Receptor Controls Cell Stemness in Acute Myeloid Leukemia and in Normal Bone Marrow

Cell Reports ◽

10.1016/j.celrep.2019.12.055 ◽

2020 ◽

Vol 30 (3) ◽

pp. 739-754.e4 ◽

Cited By ~ 8

Author(s):

Etienne Paubelle ◽

Florence Zylbersztejn ◽

Thiago Trovati Maciel ◽

Caroline Carvalho ◽

Annalisa Mupo ◽

...

Keyword(s):

Bone Marrow ◽

Vitamin D ◽

Acute Myeloid Leukemia ◽

Vitamin D Receptor ◽

Myeloid Leukemia ◽

Normal Bone Marrow ◽

Normal Bone ◽

Acute Myeloid

Download Full-text

Effect of the nonpeptide thrombopoietin receptor agonist Eltrombopag on bone marrow cells from patients with acute myeloid leukemia and myelodysplastic syndrome

Blood ◽

10.1182/blood-2009-04-219493 ◽

2009 ◽

Vol 114 (18) ◽

pp. 3899-3908 ◽

Cited By ~ 85

Author(s):

Britta Will ◽

Masahiro Kawahara ◽

Julia P. Luciano ◽

Ingmar Bruns ◽

Samir Parekh ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Receptor Agonist ◽

Mononuclear Cells ◽

Bone Marrow Mononuclear Cells ◽

Self Renewal ◽

Thrombopoietin Receptor ◽

Thrombopoietin Receptor Agonist ◽

Acute Myeloid

Abstract Thrombocytopenia is a frequent symptom and clinical challenge in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Eltrombopag is a small molecule thrombopoietin receptor agonist that might be a new option to treat thrombocytopenia in these diseases, provided that it does not stimulate malignant hematopoiesis. In this work, we studied the effects of Eltrombopag on proliferation, apoptosis, differentiation, colony formation, and malignant self-renewal of bone marrow mononuclear cells of patients with AML and MDS. Malignant bone marrow mononuclear cells did not show increased proliferation, or increased clonogenic capacity at concentrations of Eltrombopag ranging from 0.1 to 30 μg/mL. On the contrary, we observed a moderate, statistically nonsignificant (P = .18), decrease of numbers of malignant cells (mean, 56%; SD, 28%). Eltrombopag neither led to increased 5-bromo-2-deoxyuridine incorporation, decreased apoptosis, an increase of malignant self-renewal, nor enhanced in vivo engraftment in xenotransplantations. Furthermore, we found that Eltrombopag was capable of increasing megakaryocytic differentiation and formation of normal megakaryocytic colonies in patients with AML and MDS. These results provide a preclinical rationale for further testing of Eltrombopag for treatment of thrombocytopenia in AML and MDS.

Download Full-text

Hypoxia-Inducible Factor-1 Alpha (HIF-1α) and HIF-2α Are Independent Prognostic Factors for Normal Karyotype Acute Myeloid Leukemia (NK AML)

Blood ◽

10.1182/blood.v112.11.2553.2553 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2553-2553

Author(s):

Kellie M. Demock ◽

Joseph Marinaro ◽

Ian McInnis ◽

Laurie Ann Ford ◽

Meir Wetzler ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Hypoxia Inducible Factor ◽

Normal Karyotype ◽

Mrna Levels ◽

Normal Bone Marrow ◽

Normal Bone ◽

Acute Myeloid

Abstract Emerging data has shown that severe hypoxia in vitro selects for highly immature or therapy-resistant leukemia clones and may be a crucial component of leukemia stem cell niches. One mechanism utilized by normal cells to survive hypoxia is upregulation of hypoxia inducible factor-1α (HIF-1α), a master transcription factor that directly transactivates genes important for cellular responses to hypoxia including angiogenesis and anaerobic metabolic pathways. HIF-1α is rapidly degraded under normoxia but is stabilized and synthesized under hypoxia or following malignant transformation. Overexpression of HIF-1α protein is associated with increased patient (pt) mortality in multiple solid cancer types, and with worse clinical outcome in pediatric acute lymphoblastic leukemia; however, its role in acute myeloid leukemia (AML) is unknown. First, we examined the response of human AML cells in vitro to hypoxic stress. We found that in vitro exposure of human AML cell lines (HEL, HL60/VCR) with low baseline HIF-1α levels to hypoxia (≤1% O2, 5%CO2) resulted in significantly increased HIF-1α mRNA levels after 4 hours followed by increased HIF-1α protein and elevated VEGF-A and VEGFR-1 mRNA levels peaking at 8 hours. We then examined expression of HIF-1α and a related hypoxia factor, HIF-2α, in diagnostic marrow samples from 91 consecutive AML pts (46% male, 54% female) treated at our institute from 1995–2005. As karyotype is the most important prognostic factor in AML, we examined only normal karotype AML samples associated with intermediate prognosis. Median patient age was 66 years (range 21–87). Less than half (n=46, 49%) achieved complete remission (CR) following induction chemotherapy. Median overall survival (OS) was 9.6 months. HIF-1α and HIF-2α levels were measured by Q-PCR and expressed relative to normal bone marrow controls (level of mRNA expression=1). HIF-1α protein expression was also evaluated by immunohistochemistry (IHC) and qualified as nuclear vs. cytoplasmic. We found that HIF-1α mRNA levels were consistently higher in AML cells than normal bone marrow controls (median fold change 2.78; range 0.48–22.89), although HIF-1α protein levels was increased in only a minority of samples. In contrast, HIF-2α mRNA levels were consistently lower in AML samples than normal bone marrow (median 0.14; range 0.7–0.42). Univariate analysis demonstrated that age, CR, and nuclear HIF-1α protein expression by IHC (p=0.0081) impacted OS. Significant factors for event free survival (EFS) were age, CR, and cytoplasmic HIF-1α IHC expression (p=0.0422). Multivariate analysis demonstrated that age, CR, cytoplasmic HIF-1α IHC expression (p=0.0056; HR=0.22; 95% CI=0.07–0.64) and HIF-2α mRNA expression (p=0.0101; HR=0.16; 95% CI=0.04–0.65) were independent predictors for OS. Similarly, age, CR, cytoplasmic HIF-1α IHC expression (p=0.0302; HR=0.26; 95% CI=0.08–0.88), and HIF-2α mRNA levels (p=0.0016, HR 0.08, 95% CI= 0.02–0.39) were also independent factors for EFS. Conclusions: Our results are the first to demonstrate that overexpression of HIF-1α and HIF-2α are independent prognostic factors in normal karyotype AML (constituting 40–49% of all adult AML diagnoses). Given the fact that HIF expression can be upregulated by oncogenes and tumor suppressors, additional studies examining potential correlations between HIF-1/2α expression and FLT-3/NPM-1 gene mutations in NK-AML; and in other prognostic AML subgroups (i.e. AML with recurrent or complex cytogenetics) are warranted. Based on these data, inhibition of HIF-1/2α mediated pathways with targeted agents may represent a future means to improve clinical outcome for subsets of AML patients.

Download Full-text

Diverse Roles of Hepatocyte Growth Factor (HGF) in Normal Karyotype Acute Myeloid Leukemia (AML).

Blood ◽

10.1182/blood.v114.22.3107.3107 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3107-3107

Author(s):

Ian McInnis ◽

Theresa Hahn ◽

Anasitasia Ioane ◽

Ashleigh Lamson ◽

Deepika Lal ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Growth Factor ◽

Myeloid Leukemia ◽

Normal Karyotype ◽

Normal Bone Marrow ◽

Normal Bone ◽

Hepatocyte Growth ◽

Acute Myeloid

Abstract Abstract 3107 Poster Board III-44 Prior studies have demonstrated that hepatocyte growth factor (HGF) regulates proliferation and differentiation of normal hematopoietic progenitors. HGF activity occurs primarily via interactions with the c-met receptor, a tyrosine kinase receptor found on epithelial and some cancer cells. In solid tumors, HGF/c-met interactions mediate increased neoplastic invasion, metastases, and angiogenesis. However, in vitro, HGF has also been shown to mediate anti-tumor effects in leukemia cell line models. To better elucidate the role of HGF in acute leukemogenesis, we evaluated HGF and c-met gene expression in 91 normal karyotype acute myeloid leukemia (NK-AML) patient samples previously characterized for marrow angiogenesis (CD31+ microvessels), FLT-3/NPM-1 gene mutation, and pro-angiogenic factors and receptors (specifically vascular endothelial growth factors (VEGF-A and C) and their receptors). Median patient age was 66 years (range 21-87) with 49 women and 42 men. AML disease FAB subtypes M2 (37%) and M1 (36%) subtypes were most common. Median presenting white blood cell count (WBC) was 32,000/μL (range 0.43-555,000/μL) with marrow blasts of 70.6% (range 15-95.4%). Fourteen percent presented with extramedullary disease. Median OS was 9.4 months (95% CI 6.7 to 11.5 months), with median EFS of 8 months (95% CI 5.7 to 11.5 months) for all patients. Seventy-nine patients received cytarabine and anthracycline-based induction chemotherapy with 58% (n=46) achieving complete remission (CR). Marrow aspirate samples were evaluated by quantitative real-time polymerase chain reaction with levels expressed relative to normal bone marrow controls (set =1). We found that HGF gene expression was upregulated in most primary NK-AML patient samples, with 88% expressing higher HGF than normal bone marrow. Median HGF expression in AML samples was 7.73 fold higher than normal controls. Multivariate analysis including age, complete remission, marrow blasts, extramedullary disease, and expression of other angiogenic factors and receptors as covariables, showed high HGF expression to be significantly associated with both longer overall and event-free survival. Surprisingly, HGF gene expression was found to be negatively correlated with microvessel density and NPM-1 mutation and positively correlated with the VEGF receptor neuropilin-1 (NRP-1) which has been reported to function as co-mediator of HGF activity. No association between HGF and FLT-3 ITD mutation was noted. The majority of AML samples did not express the HGF receptor, c-met, suggesting that HGF function in AML occurs primarily via paracrine interactions with surrounding vascular and stromal cells and/or HGF/NRP-1 autocrine pathways. Further analysis confirmed no significant correlation between HGF and c-met gene expression in AML samples but did demonstrate a subset of NPM-1+ HGF+ AML samples (n=7) expressing high levels of both HGF and c-met (p=0.0005, r=0.96). To confirm whether HGF/c-met autocrine interactions contributed to leukemogenesis in these cells, we treated immunodeficient mice engrafted with an HGF+ c-met+ human AML cell line (HEL) with vehicle vs. a c-met tyrosine kinase inhibitor, and noted growth inhibitory effects following c-met blockade. Conclusions HGF gene expression was an independent predictive factor for improved clinical outcome and was associated with NRP-1 expression, lower microvessel density, and NPM-1 negative status in normal karyotype AML patients. A subset of AML samples was identified with high concordant HGF and c-met expression consistent with autocrine pathways. Inhibition of HGF/c-met interactions in a preclinical AML model exerted anti-tumor effects. Additional studies of the diverse roles of HGF in myeloid leukemogenesis are warranted. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Deletion of the multidrug resistance protein MRP1 gene in acute myeloid leukemia: the impact on MRP activity

Blood ◽

10.1182/blood.v95.11.3514 ◽

2000 ◽

Vol 95 (11) ◽

pp. 3514-3519 ◽

Cited By ~ 23

Author(s):

Dorina M. van der Kolk ◽

Edo Vellenga ◽

Anneke Y. van der Veen ◽

Leonore Noordhoek ◽

Hetty Timmer-Bosscha ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Multidrug Resistance ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Multidrug Resistance Protein ◽

Normal Bone Marrow ◽

Normal Bone ◽

Resistance Protein ◽

Acute Myeloid

Abstract Deletion of the multidrug resistance gene MRP1has been demonstrated in acute myeloid leukemia (AML) patients with inversion of chromosome 16 (inv[16]). These AML patients are known to have a relatively favorable prognosis, which suggests thatMRP1 might play an important role in determining clinical outcome. This study analyzed MRP1 deletion by fluorescent in situ hybridization (FISH), with a focus on inv(16) AML patients. Functional activity of multidrug resistance protein (MRP) was studied in a flow cytometric assay with the use of the MRP substrate carboxyfluorescein (CF) and the inhibitor MK-571. MRP1, MRP2, and MRP6 messenger RNA (mRNA) expression was determined with reverse transcriptase–polymerase chain reaction (RT-PCR). The results were compared with normal bone marrow cells. MRP1deletion was detected in 7 AML patients; 2 cases showed no MRP1FISH signals, and 5 cases had 1 MRP1 signal, whereas in 4 AML patients with inv(16) no MRP1 deletions were observed. A variability in MRP activity, expressed as CF efflux–blocking by MK-571, was observed (efflux-blocking factors varied between 1.2 and 3.6); this correlated with the number of MRP1 genes (r = 0.91, P < .01). MRP activity in the AML cases was not different from normal hematopoietic cells. MRP1 mRNA was detected in patients with 1 or 2 MRP1 FISH signals, but not in patients with no MRP1 signals. MRP2 and MRP6 mRNA were expressed predominantly in AML samples with 1 MRP1 signal, whereas in normal bone marrow cells no MRP2 and MRP6 mRNA was observed. In conclusion, this study shows that MRP activity varies among inv(16) AML cases and does not differ from that in normal hematopoietic cells; this might be in part due to the up-regulation of other MRP genes.

Download Full-text