FLT3 Is Involved In Ara-C Transport By Human Equilibrative Nucleoside Transporter (hENT1) In Pediatric Acute Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3844-3844
Author(s):  
Albert Català ◽  
MarÇal Pastor-Anglada ◽  
Roberta Malatesta ◽  
Susana Rives ◽  
Montserrat Torrebadell ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Cytotoxic Action ◽  
Nucleoside Transporter ◽  
Pediatric Leukemia ◽  
Equilibrative Nucleoside Transporter ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors ◽  
Flt3 Expression

Abstract Introduction Despite the remarkable improvement in survival of children with acute leukemia in the last decades, some patients still have a poor outcome. FMS-like tyrosine Kinase 3 (FLT3) is a tyrosine-kinase receptor with a key role in hematopoiesis whose mutations and overexpression have emerged as negative prognostic biomarkers in childhood leukemia. Infant leukemias (those diagnosed at age<1 year) are a special subgroup characterized by distinctive clinico-biological features, including frequent MLL (Mixed Lineage Leukemia) gene rearrangements, high FLT3 expression and high sensitivity to cytarabine (Ara-C), but a dismal prognosis. Ara-C is a deoxycytidine analog known to be transported by the human equilibrative nucleoside transporter 1 (hENT1) across the cell membrane. Once inside the cell Ara-C is activated by deoxycitidine kinase (DCK) to finally exert its cytotoxic action. High hENT1 expression levels have been involved in Ara-C sensitivity in patients with acute leukemia. However, the mechanisms that regulate the expression of hENT1 and its activity, as well as the putative relationship between FLT3 and Ara-C transport and metabolism are scarcely known. Aim To study the role of FLT3 in the regulation of the expression and activity of the main Ara-C transporters and metabolizing enzymes (ME) in pediatric leukemia. Patients, Materials, and Methods Bone marrow samples of 56 pediatric patients diagnosed with acute leukemia in Hospital Sant Joan de Déu and 3 acute leukemia cell lines (MV4-11, SEM, K562) were used for screening of Ara-C transporters and ME expression. In all cases the patients or their parents signed an informed consent. We performed a positive selection of cases with acute lymphoblastic leukemia (ALL) with hyperdiploidy (>50 chromosomes) and of MLL-rearranged cases presenting either as ALL or acute myeloblastic leukemia (AML), as these are the subgroups with highest expression of FLT3 reported in the literature, although other leukemia subtypes were also represented. The mRNA expression levels of FLT3, nucleoside transporters (NT) (hENT1, hENT2, hCNT1, hCNT3) and Ara-C ME, dCK and CNII, were quantified using real-time PCR and analyzed with the 2-ΔΔCt method, with non-neoplastic samples used as controls for the relative quantification. Direct nucleoside and Ara-C uptake measurements were performed using [5,6-3H]-nucleosides. The role FLT3 might play in the expression of NT and ME genes as well as on the activity of Ara-C transporters in these cell lines was addressed by repressing FLT3 function with its specific inhibitor PKC412 (sold only for research purposes). Results We included 56 patients (68% males) diagnosed with acute leukemia. The median age was 5.3 years (range 0-16.2), with 3 cases of infant leukemia. Fifty cases (89%) were precursor B-ALL (24 hyperdiploid cases, 5 MLL rearranged, 3 BCR-ABL+, 4 E2A-PBX1+, 5 TEL-AML1+, 9 other subtypes), 5 cases were AML (4 MLL positive cases and one case with mutated FLT3) and one case was a T-ALL harboring FLT3 mutation. As expected, the FLT3 expression was higher in cases with ALL and MLL rearrangement and, to a less extent, in ALL with hyperdiploidy. Interestingly, a significant positive correlation was found between FLT3 and hENT1 expression (mRNA levels) with all patient samples (figure 1). hENT1 expression and cytarabine-mediated uptake was significantly repressed when MV4-11 and SEM cell lines were exposed to the FLT3 inhibitor PKC412 (figures 2 & 3) without affecting hENT2, CNT1 and CNT3 expression and activity. Conclusions Our results show a strong correlation between FLT3 and the Ara-C transporter hENT1 in pediatric leukemia patients. This observation was consistent with the in vitro evidence that FLT3 inhibition resulted in hENT1 repression and down-regulation of Ara-C uptake in leukemic derived cell lines. Taken together, our data suggest that FLT3 regulates hENT1, thereby modulating the ability of cancer cells to incorporate Ara-C and promote its cytotoxic action. As FLT3 inhibitors are currently being tested as mono or combined therapy with Ara-C in several clinical trials, based upon our observations we suggest that a better schedule design might eventually be needed when dealing with treatments involving FLT3 inhibitors and Ara-C, thereby improving the outcome of this subset of patients. Disclosures: Off Label Use: The presentation include the results of in vitro studies with the FLT3 inhibitor PKC412. This drug was only used for in vitro studies, exclusively for research purposes.

scholarly journals The FMS like Tyrosine Kinase 3 (FLT3) Is Overexpressed in a Subgroup of Multiple Myeloma Patients with Inferior Prognosis

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2341
Author(s):  
Normann Steiner ◽  
Karin Jöhrer ◽  
Selina Plewan ◽  
Andrea Brunner-Véber ◽  
Georg Göbel ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tyrosine Kinase ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Tyrosine Kinase Receptor ◽  
Bone Marrow Biopsies ◽  
Flt3 Inhibitors ◽  
Flt3 Expression

Therapy resistance remains a major challenge in the management of multiple myeloma (MM). We evaluated the expression of FLT3 tyrosine kinase receptor (FLT3, CD135) in myeloma cells as a possible clonal driver. FLT3 expression was analyzed in bone marrow biopsies of patients with monoclonal gammopathy of undetermined significance or smoldering myeloma (MGUS, SMM), newly diagnosed MM (NDMM), and relapsed/refractory MM (RRMM) by immunohistochemistry (IHC). FLT3 gene expression was analyzed by RNA sequencing (RNAseq) and real-time PCR (rt-PCR). Anti-myeloma activity of FLT3 inhibitors (midostaurin, gilteritinib) was tested in vitro on MM cell lines and primary MM cells by 3H-tymidine incorporation assays or flow cytometry. Semi-quantitative expression analysis applying a staining score (FLT3 expression IHC-score, FES, range 1–6) revealed that a high FES (>3) was associated with a significantly shorter progression-free survival (PFS) in NDMM and RRMM patients (p = 0.04). RNAseq and real-time PCR confirmed the expression of FLT3 in CD138-purified MM samples. The functional relevance of FLT3 expression was corroborated by demonstrating the in vitro anti-myeloma activity of FLT3 inhibitors on FLT3-positive MM cell lines and primary MM cells. FLT3 inhibitors might offer a new targeted therapy approach in a subgroup of MM patients displaying aberrant FLT3 signaling.

In vitro studies of a FLT3 inhibitor combined with chemotherapy: sequence of administration is important to achieve synergistic cytotoxic effects

Blood ◽  
2004 ◽  
Vol 104 (4) ◽  
pp. 1145-1150 ◽  
Author(s):  
Mark Levis ◽  
Rosalyn Pham ◽  
B. Douglas Smith ◽  
Donald Small
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Chemotherapeutic Agents ◽  
In Vitro Cytotoxicity ◽  
High Risk Group ◽  
Cycle Arrest ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors ◽  
Cycle Dependent

AbstractPatients with acute myeloid leukemia (AML) harboring internal tandem duplication mutations of the FLT3 receptor (FLT3/ITD mutations) have a poor prognosis compared to patients lacking such mutations. Incorporation of FLT3 inhibitors into existing chemotherapeutic regimens has the potential to improve clinical outcomes in this high-risk group of patients. CEP-701, an indolocarbazole-derived selective FLT3 inhibitor, potently induces apoptosis in FLT3/ITD-expressing cell lines and primary leukemic blasts. We conducted a series of in vitro cytotoxicity experiments combining CEP-701 with chemotherapy using the FLT3/ITD-expressing cell lines MV4-11 and BaF3/ITD as well as a primary blast sample from a patient with AML harboring a FLT3/ITD mutation. CEP-701 induced cytotoxicity in a synergistic fashion with cytarabine, daunorubicin, mitoxantrone, or etoposide if used simultaneously or immediately following exposure to the chemotherapeutic agent. In contrast, the combination of pretreatment with CEP-701 followed by chemotherapy was generally antagonistic, particularly with the more cell cycle-dependent agents such as cytarabine. This effect appears to be due to CEP-701 causing cell cycle arrest. We conclude that in FLT3/ITD-expressing leukemia cells, CEP-701 is synergistic with standard AML chemotherapeutic agents, but only if used simultaneously with or immediately following the chemotherapy. These results should be considered when designing trials combining chemotherapy with each of the FLT3 inhibitors currently in clinical development. (Blood. 2004; 104:1145-1150)

High Level Expression of Wild Type FLT3 Is Associated with Poor Outcome and Selective Sensitivity to FLT3 Inhibitors in Childhood Acute Myeloid Leukemia: A Children’s Oncology Group Study

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 147-147
Author(s):  
Patrick Brown ◽  
Todd Alonzo ◽  
Robert Gerbing ◽  
Emily McIntyre ◽  
Beverly Lange ◽  
...  
Keyword(s):  
Wild Type ◽  
High Level Expression ◽  
High Group ◽  
Flt3 Inhibitor ◽  
Flt3 Mutations ◽  
Flt3 Inhibitors ◽  
Flt3 Expression ◽  
High Level ◽  
Risk Of Relapse

Abstract In AML, molecular prognostic markers (FLT3/ITD, WT1, NPM1, CEBPA, BAALC, e.g.) are increasingly used to complement classical cytogenetics in risk stratification. Perhaps the most important of these markers is FLT3/ITD, since it has profound prognostic significance and is pharmacologically targetable. In our prior studies using FLT3 inhibitors in both AML and ALL, we have seen samples demonstrating exquisite cytotoxic sensitivity despite the lack of FLT3 mutations. In these cases, we have shown the sensitivity to be due to high level expression of activated wild type (wt) FLT3 protein. Small studies in adult AML have suggested that high level wtFLT3 transcript expression may predict inferior clinical outcome. We hypothesized that quantitative expression of wtFLT3 may contribute to risk stratification in AML, and may identify patients that could benefit from treatment with FLT3 inhibitors. We examined diagnostic marrow samples from a cohort of 254 children with AML treated on CCG-2961. The samples were classified by FLT3 genotype (wtFLT3: N=216; FLT3/ITD, N=19, FLT3/ALM, N=19). We used qRT-PCR to determine the FLT3 expression level of the 254 samples and 10 normal bone marrow controls (NBM). Patients with FLT3 mutations had significantly higher FLT3 expression (median 10.9 fold NBM) than wtFLT3 patients (4.6 fold NBM, p&lt;0.0001). Within wtFLT3 patients, FLT3 expression was highly variable, ranging from 0.003 to 95 fold NBM. A tight correlation (r=0.85) of FLT3 expression at the RNA and protein level was observed, with FLT3 protein expression measured by FACS after staining with PE-conjugated CD135 antibodies. We grouped the eligible patients into wtFLT3 expression quartiles and analyzed outcome from study entry (N=191) and from end Course 1 for patients in CR (N=151). There were no significant differences in known covariates (gender, age, race, WBC or cytogenetics) between the highest quartile and the lower quartiles. While there was no difference in induction CR rate (p=0.54) or OS from study entry (p=0.795) between the quartiles, the highest quartile (&gt; 11.25 fold NBM, N=40) had an OS from the end Course 1 of 48 ± 18% compared to 71 ± 9% for the lower 3 quartiles (N=111, p=0.057). Various expression thresholds (from 10 fold NBM to 20 fold NBM in increments of 2 fold) were then examined. Hazard ratios (HR) for relapse and DFS were found to increase with each incremental increase in expression threshold. Using an expression threshold of 18 fold NBM, the HR for relapse and DFS were 2.3 (95% CI 1.2 to 4.7, p = 0.019) and 1.9 (95% CI 1.0 to 3.5, p = 0.042), respectively, for patients above the 18 fold threshold (N=20) vs. those below (N=131). Since FLT3/ITD is known to be a powerful predictor of poor outcome, we compared the outcome for wt FLT3 patients above the 18 fold NBM threshold with the FLT3/ITD patients treated on the CCG-2961 trial (N=53). Remarkably, the cumulative risk of relapse (60%) and DFS (30%) were essentially identical for these two groups. Subsets of wt FLT3 patients from Low (N=13), Mid (N=11) and High (N=12) level expression groups with specimens remaining in the cell bank were randomly selected for in vitro MTT cytotoxicity testing with the selective small molecule FLT3 kinase inhibitor lestaurtinib over a dose range of 5 nM to 100 nM. The mean cytotoxic response at all doses was greatest in the High group, least in the Low group, and intermediate in the Mid group. At 50 nM lestaurtinib the cytotoxic responses were 56 ± 9%, 38 ± 7% and 21 ± 8% in the High, Mid and Low groups, respectively (p&lt;0.0001). Remarkably, the cytotoxic response for the High group (56%) was similar to that of fifteen FLT3/ITD+ samples we previously tested under identical conditions (48%, published in Blood104(6):1841). In conclusion, FLT3 mRNA and protein expression varies widely among patients with wt FLT3, and patients with the highest levels of wt FLT3 expression have a significantly increased risk of relapse and death. Furthermore, the leukemic blasts from these patients are exquisitely and selectively sensitive to FLT3 inhibition in vitro. Remarkably, high wt FLT3 expression was indistinguishable from FLT3/ITD in its strength as a poor prognostic factor and as a predictor of FLT3 inhibitor sensitivity. These data suggest that prospectively determining high level expression of wt FLT3 may be useful not only in identifying a high risk group, but also in selecting a group of patients for whom FLT3 inhibitor therapy may be indicated.

scholarly journals FLT3 Inhibitor Treatment Increases FLT3 Expression That Exposes FLT3-ITD+ AML Blasts to Elimination By FLT3 CAR-T Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 903-903 ◽  
Author(s):  
Hardikkumar Jetani ◽  
Irene García-Cadenas ◽  
Thomas Nerreter ◽  
Ralph Goetz ◽  
Jorge Sierra ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Lines ◽  
Combination Treatment ◽  
Car T Cells ◽  
Flt3 Inhibitor ◽  
Car T ◽  
Flt3 Inhibitors ◽  
Flt3 Expression ◽  
Inhibitor Treatment

Abstract Background: FMS-like tyrosine kinase 3 (FLT3) is a transmembrane protein uniformly expressed on leukemic blasts in acute myeloid leukemia (AML), and driver of leukemia-genesis in FLT3-ITD+ (Internal tandem duplication) AML. There is an increasing body of pre-clinical and clinical data suggesting that FLT3-ITD+ AML blasts respond to FLT3 inhibitor treatment by augmenting FLT3 expression in order to sustain the survival signal provided by this mutation. Here, we analyzed FLT3 expression on FLT3 wild type and FLT3-ITD+ AML cells after treatment with the FLT3 inhibitors midostaurin, quizartinib and crenolanib, and determined the antileukemia efficacy of combination treatment with FLT3 inhibitors and FLT3 CAR T cells in vitro and in vivo. Methods: MOLM-13 and MV4;11 AML cells (both FLT3-ITD+) were cultured in the presence of IC50 doses of midostaurin, quizartinib and crenolanib, respectively to induce resistance (MOLM-13R/MV4;11R). A FLT3-CAR comprised of BV10 scFv binding domain, CD28-CD3ζ signal module and EGFRt marker was encoded in a lentiviral vector and expressed in CD8+ and CD4+ T cells of healthy donors and patients (n=6). T cell mediated cytolytic activity was evaluated in luminescence-based assay, cytokine production analyzed by ELISA and proliferation assessed by CFSE dye dilution. NSG mice (n= 4-6 per group) were engrafted with MOLM-13/ffLuc AML cells and treated with 5x106 CAR T cells alone or in combination with FLT3 inhibitors. Results: We detected a significant increase in FLT3 expression on both MV4;11 and MOLM-13 AML cells after treatment with each of the inhibitors as assessed by mean fluorescence intensity (quizartinib > crenolanib > midostaurin). The increase in FLT3 expression occurred specifically on these FLT3-ITD+ AML cell lines and was not observed on FLT3 wt AML (THP-1), acute lymphoblastic leukemia (NALM-16), mixed lineage leukemia (KOPN-8 and SEM) cell lines and normal hematopoietic stem cells. We applied single molecule sensitive super-resolution microscopy to demonstrate that the average number of FLT3 molecules (per micrometer sq.) on MV4;11 AML cells had increased from 0.80 (untreated) to 10.7 (quizartinib), 4.7 (crenolanib), and 3.3 (midostaurin) (p<.05). Of interest, midostaurin induced clustering of FLT3, while FLT3 was still present as monomers after quizartinib and crenolanib treatment. Intriguingly, the higher FLT3 density after FLT3 inhibitor treatment translated into superior antileukemia reactivity of FLT3 CAR T cells against AML cell lines and primary AML cells in vitro and in vivo. We observed the strongest increase in cytolytic activity, cytokine production and proliferation by CD8+ and CD4+ FLT3 CAR T cells after treatment with crenolanib and quizartinib, followed by midostaurin (p<.05). We confirmed that upregulation of FLT3 occurred on MOLM-13 cells during FLT3 inhibitor therapy in NSG mice in vivo, and observed synergistic antileukemia efficacy of FLT3 CAR T cells in combination with each of the compounds. The mean frequency of FLT3 CAR T cells in mice that received FLT3 CAR T cells and an FLT3 inhibitor was 2-4 fold higher compared to mice had received FLT3 CAR T cells alone (p<.05) and was the highest in the cohort of mice that had received FLT3 CAR T cells in combination with crenolanib. FLT3 CAR T cells alone and each of the combination treatments of FLT3 CAR T & FLT3 inhibitor achieved 100% response rate which compares favorably to untreated or FLT3 inhibitor alone (0%). However, the mean fold reduction in leukemia burden (b/w day 7 and 10) was greater in all three combination treatment compare to only CAR treatment (p<.05). The most potent combination was FLT3 CAR T cells & crenolanib that accomplished the strongest reduction in leukemia burden as assessed by bioluminescence imaging and flow cytometry. Conclusion: Collectively, the data show that FLT3 inhibitors augment cell surface expression of FLT3 in FLT3-ITD+ AML cells which leads to enhanced recognition and elimination by FLT3 CAR T cells. This is, to our knowledge, the first demonstration that small molecule inhibitors and CAR T cell immunotherapy can be used synergistically to treat a hematologic malignancy. We confirmed this principle with each of the FLT3 inhibitors in our panel, and observed the strongest antileukemia activity of FLT3 CAR T cells in combination with crenolanib. Our data encourage the clinical evaluation of this combination treatment in high risk patients with FLT3-ITD+ AML. Disclosures Jetani: University hospital wuerzburg: Employment, Patents & Royalties: H.J. and M.H are co-inventors on a patent related to the use of FLT3-CAR T-cells to treat AML filed by the University of Wuerzburg, Wuerzburg, Germany. Bonig:Kiadis Pharma: Consultancy.

scholarly journals ATM/G6PD-driven redox metabolism promotes FLT3 inhibitor resistance in acute myeloid leukemia

2016 ◽  
Vol 113 (43) ◽  
pp. E6669-E6678 ◽  
Author(s):  
Mark A. Gregory ◽  
Angelo D’Alessandro ◽  
Francesca Alvarez-Calderon ◽  
Jihye Kim ◽  
Travis Nemkov ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Mitochondrial Oxidative Stress ◽  
Flt3 Inhibitor ◽  
A Genome ◽  
Flt3 Inhibitors ◽  
Acute Myeloid

Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are common in acute myeloid leukemia (AML) and drive leukemic cell growth and survival. Although FLT3 inhibitors have shown considerable promise for the treatment of AML, they ultimately fail to achieve long-term remissions as monotherapy. To identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a genome-wide RNA interference (RNAi)-based screen that identified ATM (ataxia telangiectasia mutated) as being synthetic lethal with FLT3 inhibitor therapy. We found that inactivating ATM or its downstream effector glucose 6-phosphate dehydrogenase (G6PD) sensitizes AML cells to FLT3 inhibitor induced apoptosis. Examination of the cellular metabolome showed that FLT3 inhibition by itself causes profound alterations in central carbon metabolism, resulting in impaired production of the antioxidant factor glutathione, which was further impaired by ATM or G6PD inactivation. Moreover, FLT3 inhibition elicited severe mitochondrial oxidative stress that is causative in apoptosis and is exacerbated by ATM/G6PD inhibition. The use of an agent that intensifies mitochondrial oxidative stress in combination with a FLT3 inhibitor augmented elimination of AML cells in vitro and in vivo, revealing a therapeutic strategy for the improved treatment of FLT3 mutated AML.

scholarly journals HIF-1–dependent repression of equilibrative nucleoside transporter (ENT) in hypoxia

2005 ◽  
Vol 202 (11) ◽  
pp. 1493-1505 ◽  
Author(s):  
Holger K. Eltzschig ◽  
Parween Abdulla ◽  
Edgar Hoffman ◽  
Kathryn E. Hamilton ◽  
Dionne Daniels ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Molecular Mechanisms ◽  
Hypoxia Inducible Factor ◽  
Nucleoside Transporter ◽  
In Vivo Models ◽  
Equilibrative Nucleoside Transporter ◽  
Hypoxia Inducible Factor 1 ◽  
And Function

Extracellular adenosine (Ado) has been implicated as central signaling molecule during conditions of limited oxygen availability (hypoxia), regulating physiologic outcomes as diverse as vascular leak, leukocyte activation, and accumulation. Presently, the molecular mechanisms that elevate extracellular Ado during hypoxia are unclear. In the present study, we pursued the hypothesis that diminished uptake of Ado effectively enhances extracellular Ado signaling. Initial studies indicated that the half-life of Ado was increased by as much as fivefold after exposure of endothelia to hypoxia. Examination of expressional levels of the equilibrative nucleoside transporter (ENT)1 and ENT2 revealed a transcriptionally dependent decrease in mRNA, protein, and function in endothelia and epithelia. Examination of the ENT1 promoter identified a hypoxia inducible factor 1 (HIF-1)–dependent repression of ENT1 during hypoxia. Using in vitro and in vivo models of Ado signaling, we revealed that decreased Ado uptake promotes vascular barrier and dampens neutrophil tissue accumulation during hypoxia. Moreover, epithelial Hif1α mutant animals displayed increased epithelial ENT1 expression. Together, these results identify transcriptional repression of ENT as an innate mechanism to elevate extracellular Ado during hypoxia.

Identification and Effects of Novel Promoter Region Haplotypes in the Human Equilibrative Nucleoside Transporter, hENT1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2083-2083
Author(s):  
Scott N. Myers ◽  
Rakesh K. Goyal ◽  
Jennifer D. Roy ◽  
Robert E. Ferrell
Keyword(s):  
De Novo ◽  
Remission Rate ◽  
Leukemic Cells ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Nucleoside Transporter ◽  
Nucleotide Polymorphisms ◽  
Flanking Sequence ◽  
Equilibrative Nucleoside Transporter

Abstract Front-line induction chemotherapy regimens containing cytosine arabinoside (Ara-C) and anthracyclines result in 80% complete remission rate in childhood acute myeloid leukemia (AML) but their cure rate is about 35 – 50%, one of the lowest of all childhood cancers. Understanding the factors that contribute to emergence of chemoresistant leukemic cells is crucial to improving treatment outcome in children with AML. We are interested in studying the role of variation in Ara-C transport and biotransformation pathway genes in the efficacy and toxicity of treatment of childhood AML. To permeate the cell membrane, Ara-C is mainly dependent on human equilibrative nucleoside transporter 1 (hENT1; SLC29A1; gene localized to 6p21.1). Several studies have suggested an important role for altered levels of hENT1 in the chemosensitivity of AML blasts to Ara-C (Galmarini et al. Leukemia2001; 15(6):87; Gati et al. Leuk Lymphoma1998; 32(1–2):45). Osato and colleagues identified two single nucleotide polymorphisms (SNPs) in the hENT1 coding sequence that led to missense changes, but their in vitro analysis did not detect differences in the activity of variant alleles in a yeast transfection system (Osato et al. Pharmacogenetics2003;13(5):297). To identify variation in hENT1 that might influence its expression, we sequenced 1.6Kb of the proximal 5′-flanking sequence of the gene in 42 unrelated individuals and identified three SNPs at positions C-1345G, G-1050A, and G-706C. TRANSFAC analysis (www.genomatix.de) predicted that two of these (C-1345G & G-706C) would alter consensus transcription factor binding site sequences. We cloned four naturally occurring haplotypes (CGG, CAG, CGC, and GAG) using the TOPO-TA cloning kit, then transfected Cos-1 cells using the Lipofectamine 2000 protocol. Gene expression was assayed using the Promega Dual-Luciferase Reporter Assay System and read on a Molecular Devices HT Analyzer. Luciferase activity was measured at 24 and 48 hours after transfection for six replicates of every condition during three separate transfections. To correct for differences in transfection efficiencies, experimental (Photinus pyralis) luciferase activities were normalized by co-transfection with control (Renilla reniformis) luciferase plasmid. Compared to the wild type CGG haplotype, variant haplotypes CAG, CGC, and GAG drive luciferase expression at approximately 2x (p <0.0001), 1.4x (p <0.001) and 1.2x (p =0.08), respectively. This leads to the hypothesis that individuals carrying CAG or CGC haplotypes (17% of the population) exhibit higher levels of hENT1 expression and are more sensitive to Ara-C exposure. Experiments are underway to quantify gene transcripts in people of known hENT1 haplotypes. We also plan to genotype a large cohort of children with de novo AML for these three SNPs in hENT1 and correlate clinical outcomes in individuals carrying the low- versus the high-expressing haplotypes.

Lack of BIC and microRNA miR-155 Expression in Primary Cases of Burkitt Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1922-1922
Author(s):  
Joost Kluiver ◽  
Eugenia Haralambieva ◽  
Debora de Jong ◽  
Tjasso Blokzijl ◽  
Susan Jacobs ◽  
...  
Keyword(s):  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Burkitt Lymphoma ◽  
High Expression ◽  
Common Finding ◽  
Type I ◽  
Pediatric Leukemia ◽  
Normal Peripheral Blood ◽  
Type Iii

Abstract We previously demonstrated a high expression of primary-microRNA BIC (pri-miRNA-155) in Hodgkin lymphoma (HL) and lack of expression in most non-Hodgkin lymphoma subtypes including some Burkitt lymphoma (BL) cases. Recently, a high expression of BIC was reported in BL in comparison to pediatric leukemia and normal peripheral blood samples. In this study we extended our series of BL cases and cell lines for BIC expression by RNA in-situ hybridization (ISH) and quantitative (q)RT-PCR. Both BIC RNA-ISH and qRT-PCR revealed no or only low levels of BIC in 25 BL tissues, including 7 Epstein-Barr virus (EBV) positive cases, compared to HL and normal controls. In agreement with these findings, Northern blotting revealed absence of miR-155 in BL tissues. EBV negative and EBV latency type I BL cell lines also showed very low BIC and miR-155 expression levels as compared to HL cell lines. Higher levels of BIC and miR-155 were detected in in vitro transformed lymphoblastoid EBV latency type III BL cell lines. An association of latency type III infection and induction of BIC was supported by consistent expression of BIC in 11 and miR-155 in 2 posttransplantation lymphoproliferative disorder (PTLD) cases. In summary, we demonstrated that expression of BIC and miR-155 is not a common finding in BL. Expression of BIC and miR-155 in 3 latency type III EBV positive BL cell lines and in all primary PTLD cases suggests a possible role for EBV latency type III specific proteins in the induction of BIC expression.

Heterogeneity in Phenotype, Functional Capacity, and Drug Sensitivity for Pediatric Acute Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2654-2654
Author(s):  
Christina M. Wiedl ◽  
Terzah M. Horton ◽  
Randall M Rossi ◽  
Sarah J Neering ◽  
Valerie Grose ◽  
...  
Keyword(s):  
Stem Cell ◽  
High Risk ◽  
Acute Leukemia ◽  
Aldehyde Dehydrogenase ◽  
Pediatric Leukemia ◽  
Treatment Regimens ◽  
Resistant Population ◽  
Drug Studies ◽  
Selection Of

Abstract Abstract 2654 Poster Board II-630 Introduction: With the institution of multidrug, multiphase chemotherapy regimens, major improvements in clinical outcomes have been made in pediatric acute leukemia patients in the last thirty years. However, there remains a substantial percentage of pediatric patients who relapse and die of their disease, particularly with high risk ALL, T cell ALL and AML. It is possible that these patients' disease initiates from a leukemic stem cell such as those found in adult myeloid disease, or at the very least, harbor a chemo-resistant population. Our research has two main aims: first to evaluate the functional and phenotypic heterogeneity within standard risk (SR), high risk (HR) and relapsed (RD) pediatric leukemia. Second, to evaluate current treatment regimens for the selection of a chemo-resistant or LSC populations and then attempt to target this population with novel treatments. Methods: In vitro studies for functional heterogeneity include colony-forming assays (CFU) using methylcellulose and limiting-dilution suspension culture studies. Phenotypic heterogeneity is evaluated with multi-color flow cytometry and detection of alterations in aldehyde dehydrogenase activity. Xenograft studies in immune deficient mice are used to evaluate self-renewal capability, serial engraftment kinetics, and alterations in phenotype. Drug studies are performed by evaluating the differences in phenotype and CFU over time when treating with conventional induction chemotherapy or novel agents. Results: We have evaluated several SR and HR ALL samples in addition to some RD samples, which are paired with HR diagnostic samples. In vitro studies revealed the SR samples had little to no colony forming ability (0-1%) while the HR samples had approximately 3-5% and the RD samples 8-10% colony-forming ability. Likewise, the SR samples failed to engraft NOD-SCID mice while the HR samples, from patients with infantile ALL and the MLL translocation or T cell ALL, had robust engraftment in primary and secondary recipients. The engraftment kinetics were uniformly faster in secondary recipients. These findings suggest that HR leukemia may be the result of a leukemia-initiating cell with stem cell-like characteristics while SR ALL may arise from a more committed lymphoid progenitor. Interestingly, in the RD samples, several of the phenotypic markers are similar to that of the primary sample after treatment with induction therapy, particularly with regards to percentages of CD 34, 133-1, 133-2 and aldehyde dehydrogenase levels. Several HR samples have been exposed to induction chemotherapy (Decadron, Cytarabine, Doxorubicin and Vincristine), and the CFU potential and phenotype evaluated over a two-week time course. Notably, the majority of bulk disease is effectively killed, the CFU content actually increases two to three-fold, when an equivalent number of viable cells are analyzed. Furthermore, the phenotype reveals brighter staining with several proposed stem cell markers (CD34, 117, 133-1, 133-2, 123, and measurement of aldehyde dehyrogenase). These data indicate the selection of a chemo-resistant or LSC population. Conclusions: Our results to this point suggest important differences both functionally and phenotypically, between SR, HR and RD pediatric leukemia. These findings are consistent with what would be expected given clinical differences in each of these disease states and begins to establish a means of identifying a LSC or chemo-resistant population, which can be targeted with novel treatment regimens. Likewise, these techniques may also provide a means of evaluating for minimal residual disease (MRD) in a LSC or chemo-resistant population by identifying that population's phenotype by passaging the initial sample through serial murine engraftments or in vitro drug studies. Disclosures: No relevant conflicts of interest to declare.

Aurora Kinases in Childhood Acute Leukemia: The Promise of Aurora Kinase B As Drugable Target

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1476-1476
Author(s):  
Stefanie A. Segers ◽  
C. Michel Zwaan ◽  
Carla Exalto ◽  
Mirjam W.J. Luijendijk ◽  
Valerie S. Calvert ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Aurora Kinase ◽  
Selective Inhibitor ◽  
Leukemic Cells ◽  
Aurora Kinases ◽  
Aurora Kinase B ◽  
Pediatric All ◽  
Childhood Acute Leukemia

Abstract Abstract 1476 AIM: Aurora kinases (AURK) A and B are known regulators of mitosis and are overexpressed in a large number of human cancers, including leukemia. Several AURK-inhibitors have shown anti-tumor activity in vitro and in vivo. However, the efficacy of AURK inhibition in the treatment of childhood acute leukemia is unexplored. We therefore investigated the effect of targeting AURKA and AURKB in leukemic cells of children with newly diagnosed acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Materials & Methods: Affymetrix gene expression data of 297 ALL, 237 AML and 8 normal bone marrow (nBM) samples were analyzed for AURKA and B mRNA expression levels. Protein expression levels in 172 pediatric ALL and 10 nBM samples were determined with a reverse phase protein array. Functional studies were performed in ALL and AML cell lines, in which AURKA and B were silenced using a short hairpin RNA with a lentiviral delivery system or LNA-containing oligonucleotides. Sensitivity of leukemic cell lines to the AURKB-selective inhibitor Barasertib-hQPA (AZD1152-hQPA) was tested in vitro with an MTS assay. Results: AURKA and B mRNA levels were low in ALL and AML patients. In contrast, Aurora A and B proteins were expressed to a greater extent in patients (p<0.0002), especially in ALL cases with an E2A-PBX1 translocation (p<0.0001) than in nBM mononuclear cells. Silencing of AURKA by shRNA and by LNA-oligonucleotide caused no or only minor growth delay in several cell lines reflecting genetic subtypes typically found in pediatric ALL and AML. In contrast, silencing of AURKB resulted in proliferation arrest and apoptosis in these cells. Furthermore, 18 out of 20 ALL and AML cell lines tested were highly sensitive to the AURKB-selective inhibitor Barasertib-hQPA in the nanomolar range (IC50 = 19–233 nM) whereas less sensitivity was seen for other inhibitors. Conclusion: These data show that inhibition of AURKB but not AURKA has an anti-proliferative and pro-apoptotic effect on acute leukemic cells. Thus, targeting Aurora Kinase B may offer a new strategy to treat pediatric ALL and AML. Disclosures: No relevant conflicts of interest to declare.

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