Specific Inhibition of BCR-ABL Gene Expression in Acute Lymphoblastic Leukemia by Catalytic DNAzymes.

Abstract The BCR-ABL fusion protein p190 resulting from the translocation t(9;22) exhibits dysregulated tyrosine kinase activity and was shown to cause acute lymphoblastic leukemia (ALL). Detection of the BCR-ABL fusion gene in childhood ALL is associated with an adverse prognosis and defines a group of high risk patients. Because the BCR-ABL gene fusion is specific for leukemic cells it represents an ideal target for leukemia specific treatment approaches. Catalytic DNAzymes are able to cleave mRNA in a sequence specific manner, causing inhibition of protein translation from the DNAzyme targeted mRNA both in vitro and in vivo. In order to cut off the BCR-ABL driven malignant proliferation, we designed DNAzymes to impede the expression of p190 BCR-ABL by cleaving the BCR-ABL mRNA adjacent to the fusion site. One construct was found that cleaved the target mRNA efficiently and specifically leaving BCR and ABL, relevant for normal cell survival and proliferation, unaffected. Activity and specificity of the BCR-ABL DNAzyme was investigated in cleavage assays with in vitro transcribed BCR-ABL, BCR and ABL mRNA. DNAzymes were delivered to cultured BCR-ABL+ ALL cells by lipid transfection. The efficiency of cellular delivery reached 90% as studied by flow cytometry, fluorescence microscopy and confocal microscopy after transfection of FITC labeled DNAzymes. To control for unspecific effects of DNAzyme delivery as well as for antisense effects, a catalytically inactive DNAzyme still exhibiting BCR-ABL antisense activity was designed. Fourty-eight hours after a single treatment of BCR-ABL+ ALL-cells with DNAyzmes the BCR-ABL mRNA concentration, as measured by quantitative real-time RT-PCR, was significantly reduced by 56% and 66% compared to controls treated with the inactivated DNAzyme and to untreated cells, respectively. Western blot analysis showed a decrease in p190 protein levels after DNAzyme treatment in comparison to the control treated with inactive DNAzyme as well as to the untreated cells. Most noteworthy, four days after a single DNAzyme treatment the net growth of BCR-ABL+ ALL cells treated with the active DNAzyme was inhibited by 68% compared to the untreated control. From these data we conclude, firstly, DNAzymes targeting mRNA coding for the minor BCR-ABL variant are able to significantly reduce the amount of fusion mRNA in the cells, leading to a reduction in protein expression, followed by the inhibition of BCR-ABL driven proliferation of ALL cells. Secondly, this exemplified setting gives a hint that DNAzymes might be of therapeutic use in hematopoietic malignancies associated with specific mutations, expressing oncogenic fusion genes or overexpressing oncogenic genes.

Download Full-text

Pharmacokinetic and Pharmacodynamic Effects of PEG-Asparaginase in Newly Diagnosed Childhood Acute Lymphoblastic Leukemia: Results from a Single Agent Window Study.

Blood ◽

10.1182/blood.v110.11.860.860 ◽

2007 ◽

Vol 110 (11) ◽

pp. 860-860

Author(s):

Inge M. Appel ◽

Karin M. Kazemier ◽

Anjo J.P. Veerman ◽

Elisabeth van Wering ◽

Monique L. Den Boer ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Clinical Response ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Hazard Ratio ◽

Leukemic Cells ◽

Newly Diagnosed ◽

Pharmacodynamic Effects

Abstract L-Asparaginase is an effective drug for treatment of children with acute lymphoblastic leukemia. The effectiveness is generally thought to result from a rapid depletion of asparagine in serum and cells. Several studies have shown that in vitro resistance to this drug is an independent prognostic factor in ALL. We investigated the clinical response of one in vivo dose of 1000 IU/m2 PEG-Asparaginase and its pharmacokinetic and pharmacodynamic effects in children with newly diagnosed ALL before the start of combination chemotherapy. 57 children (36M / 21F) were enrolled in the study: 2 pro B-ALL, 38 common/ pre B-ALL and 17 T-ALL. Genotyping of precursor B-ALL revealed 11 hyperdiploid, 8 TELAML1 positive, 2 BCRABL positive, no MLL rearrangement, 8 normal, 11 others. The clinical response to PEG-Asparaginase on day 0 (5 days after the PEG-Asparaginase infusion) was defined as good when the number of leukemic cells of peripheral blood was < 1 × 109/L, as intermediate when leukemic cells were 1-10 × 109/L, and as poor when leukemic cells were > 10 × 109/L. The in vivo window response was significantly related to immunophenotype and genotype: 26/38 common / pre B-ALL cases, especially those with hyperdiploidy and TELAML1 rearrangement, demonstrated a good clinical response compared to 8/17 T-ALL (p=0.01). Both BCRABL positive ALL cases showed a poor response (p=0.04). A poor in vivo clinical window response was related to in vitro resistance to L-Asparaginase (p=0.02) and both in vitro as well as in vivo response were prognostic factors for long-term event-free survival (Hazard ratio 6.4; p=0.004, and Hazard ratio 3.7; p=0.01, respectively). The L-Asparaginase activity in the serum was >100 IU/L for at least 15 days. The asparagine levels remained below the detection limit of 0.2 mM for at least 26 days with a concomitant rise in serum aspartate and glutamate. These findings confirm that PEG-Asparaginase will yield its pharmacodynamic effects for 2-4 weeks. After administration of one in vivo dose of 1000 IU/m2 PEG-Asparaginase no changes in apoptotic parameters or changes in intracellular levels of twenty amino acids in leukemic cells could be measured, in contradiction to the changes found after in vitro exposure. This may be explained by the rapid removal of apoptotic cells from the circulation in vivo. Otherwise it is possible that in vivo mesenchymal cells from the bone marrow supply leukemic blasts with asparagine in response to treatment with L-Asparaginase. Conclusion: The clinical response to one dose of 1000 IU/m2 PEG-Asparaginase intravenously is related to phenotype and genotype and predicts outcome. These results suggest that children with ALL with a poor clinical response to PEG-Asparaginase might benefit from a more intensive antileukemic therapy.

Download Full-text

Association of high-level MCL-1 expression with in vitro and in vivo prednisone resistance in MLL-rearranged infant acute lymphoblastic leukemia

Blood ◽

10.1182/blood-2009-02-205963 ◽

2010 ◽

Vol 115 (5) ◽

pp. 1018-1025 ◽

Cited By ~ 49

Author(s):

Ronald W. Stam ◽

Monique L. Den Boer ◽

Pauline Schneider ◽

Jasper de Boer ◽

Jill Hagelstein ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Potential Role ◽

Pediatric Patients ◽

Lymphoblastic Leukemia ◽

Poor Response ◽

Childhood All ◽

Polymerase Chain ◽

High Level

Abstract MLL-rearranged acute lymphoblastic leukemia (ALL) represents an unfavorable type of leukemia that often is highly resistant to glucocorticoids such as prednisone and dexamethasone. Because response to prednisone largely determines clinical outcome of pediatric patients with ALL, overcoming resistance to this drug may be an important step toward improving prognosis. Here, we show how gene expression profiling identifies high-level MCL-1 expression to be associated with prednisolone resistance in MLL-rearranged infant ALL, as well as in more favorable types of childhood ALL. To validate this observation, we determined MCL-1 expression with quantitative reverse transcription–polymerase chain reaction in a cohort of MLL-rearranged infant ALL and pediatric noninfant ALL samples and confirmed that high-level MCL-1 expression is associated with prednisolone resistance in vitro. In addition, MCL-1 expression appeared to be significantly higher in MLL-rearranged infant patients who showed a poor response to prednisone in vivo compared with prednisone good responders. Finally, down-regulation of MCL-1 in prednisolone-resistant MLL-rearranged leukemia cells by RNA interference, to some extent, led to prednisolone sensitization. Collectively, our findings suggest a potential role for MCL-1 in glucocorticoid resistance in MLL-rearranged infant ALL, but at the same time strongly imply that high-level MCL-1 expression is not the sole mechanism providing resistance to these drugs.

Download Full-text

Clonal Origins of 'late' Relapses in ETV6-RUNX1 Acute Lymphoblastic Leukemia..

Blood ◽

10.1182/blood.v114.22.1583.1583 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1583-1583

Author(s):

Frederik W van Delft ◽

Sharon W Horsley ◽

Kristina Anderson ◽

Caroline M Bateman ◽

Susan Colman ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Copy Number ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Childhood All ◽

Remission Duration ◽

Clonal Origin

Abstract Abstract 1583 Poster Board I-609 Approximately a quarter of B cell precursor childhood acute lymphoblastic leukemia (ALL) is characterized by an ETV6-RUNX1 (TEL-AML1) fusion gene and has an overall good prognosis. The majority of these children will be treated on the standard risk arm of the United Kingdom ALL treatment protocols. Relapse usually occurs after cessation of treatment but remarkably can present many years later. The incidence of ETV6-RUNX1 at relapse has been reported to be less than or similar to de novo ALL. Molecular studies on neonatal bloodspots and on twins with concordant ALL have demonstrated the prenatal origin of major subtypes of childhood ALL, including most ETV6-RUNX1 fusion gene positive cases. In addition these investigations have suggested the existence of a preleukaemic stem cell requiring additional mutations or ‘hits’ in order to develop frank leukemia. To understand the genetic basis and clonal origin of late relapses we have compared the profiles of genome-wide copy number alterations (CNA) at relapse versus presentation in samples matched with remission DNA from 24 patients. The selected samples had tumor cell purity >75% before DNA extraction. DNA copy number alteration data was generated using the Affymetrix 500K SNP arrays. LOH analysis was performed using CNAG 3.0 and dCHIP 2008. Overall we identified 168 CNA at presentation and 252 at relapse (excluding deletions at IgH and TCR loci), equating to 6.96 and 10.3 CNA at presentation and relapse respectively. Although the number of CNA increased at relapse, no single gene or pathway was uniquely targeted in relapse. The most frequent alterations involved loss of 12p3.2 (ETV6), 9p21.3 (CDKN2A/B), 6q16.2-3 and gain of 21q22.1-22.12. A novel observation was gain of part or whole of chromosome 16 (2 patients at presentation, 5 at relapse) and deletion of the oncogene Plasmocytoma Variant Translocation 1 (PVT1) in 3 patients. Pathway analysis demonstrated frequent involvement at presentation and relapse of genes implicated in both B cell development (44 versus 46%) and cell cycle control (46 versus 71%). In order to study the clonal origin of relapse, we devised a classification describing the change in CNA between presentation and relapse in each individual patient. The clonal relationship between the presentation and relapse clone was established by the persistence of both the ETV6-RUNX1 fusion and at least 1 Ig and/or TCR rearrangement. We used a classification focussed on ‘driver’ CNA, defined as CNA that target genes functionally involved in leukemogenesis or CNA that are recurrently targeted as described in the literature. The four categories of relapse were type 1 (the dominant clone at presentation presented unchanged at relapse), type 2 (the relapse clone was derived from the major subclone at presentation with additional CNA), type 3 (the relapse clone was derived from a minor clone at presentation with gains and losses of CNA) and type 4 (the relapse clone is derived from an ancestral or preleukemic clone at initial presentation with all CNA gained). Twenty-one of the 24 patients were classifiable in this way (Figure 1). Although comparative relapse / presentation CNA profiles cannot identify precise clonal origins of relapse, the data indicate that irrespective of time to relapse (<2 to 9.9 years), the relapse clone appeared to be derived from either a major or minor clone at diagnosis with none (0/6) of the very late relapses (>5 years) derived from pre-leukemic cells lacking CNA. This data indicate diverse clonal origins of relapse and extended periods of dormancy, possibly via quiescence, for stem cells in ETV6-RUNX1+ ALL. Relapse type Remission duration (years) < 2 2 - 5 > 5 1 • • 2 • ••••••• •• 3 •• •• ••• 4 •• Figure 1. Each patient is represented by a black dot. Each patient is classified on the basis of the relapse type and remission duration. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Efficacy of Focal Adhesion Kinase Inhibition in Combination with Dasatinib in BCR-ABL1 Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v126.23.3766.3766 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3766-3766 ◽

Cited By ~ 1

Author(s):

Michelle L. Churchman ◽

Luke Jones ◽

Kathryn Evans ◽

Jennifer Richmond ◽

Irina M Shapiro ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Leukemic Cells ◽

Employment Equity ◽

Self Renewal ◽

Equity Ownership

Abstract Introduction: BCR-ABL1+ B-progenitor acute lymphoblastic leukemia (Ph+ B-ALL) is a highly aggressive disease that is often refractory to currently available therapies. Our previous genomic profiling studies have identified loss-of-function or dominant negative mutations in IKZF1, encoding the lymphoid transcription factor Ikaros, in over 80% of Ph+ ALL. In addition, deletion of CDKN2A, which encodes the INK4A and ARF tumor suppressors, is observed in approximately half of all cases (Mullighan et al., 2008). Alterations of IKZF1 are associated with poor outcome despite the use of tyrosine kinase inhibitors (TKIs). Ikzf1 alterations, including Ikaros isoform 6 (IK6), result in the acquisition of stem cell-like features, enhanced self-renewal, expression of adhesion molecules, and transcriptional upregulation of focal adhesion kinase (FAK), resulting in increased adhesion in vitro and in vivo, and decreased sensitivity to TKIs (Churchman, Cancer Cell, in press). VS-4718 is a potent, selective, and orally bioavailable FAK inhibitor currently under evaluation in a phase 1 clinical trial in subjects with various solid tumors, however in vivo efficacy in hematological malignancies had not been evaluated. Targeting FAK with VS-4718 is an attractive approach to abrogate the adhesive phenotype of IKZF1-altered leukemic cells potentially enhancing the effects of dasatinib in the treatment of high-risk BCR-ABL1 B-ALL. Methods: We examined the efficacy and mechanisms of FAK inhibition using VS-4718 as a single agent and in combination with dasatinib in vitro and in vivo in a range of xenograft and genetically engineered mouse models of BCR-ABL1 ALL. Each model had concomitant deletion of Arf which is observed in approximately 50% of human cases. Results: A pre-clinical in vivo trial of dasatinib and VS-4718 combination therapy in a murine C57Bl/6 Arf-/- BCR-ABL1 pre-B cell model resulted in a marked increase in survival in both IK6-expressing and non-IK6 cohorts of mice, and one complete long-term remission in the IK6-expressing group. Further, we showed increased efficacy of VS-4718 and dasatinib, compared to either agent alone, against two highly aggressive human Ph+ IK6-expressing B-ALL xenografts in vivo, with decreased infiltration of leukemic cells in bone marrow and spleens demonstrating a synergistic effect of the VS-4718/dasatinib combination. In vitro cell viability was reduced with induction of apoptosis at increasing concentrations of VS-4718 as a single agent, and further potentiated the effects of dasatinib in cytotoxicity assays using human xenografted and murine leukemic cells. VS-4718 profoundly diminished the ability of BCR-ABL1-expressing cells to form cell-matrix adhesions in vitro, as evident by the reduced adherence to fibronectin monolayers and bone marrow stromal cells. VS-4718 almost completely abolished the colony-forming potential of BCR-ABL1-expressing murine pre-B cells with and without Ikzf1 alterations at drug concentrations that do not affect cell viability suggestive of a reduction in self-renewal. Calvarial imaging of mice transplanted with Ikzf1-altered BCR-ABL1 leukemic cells and treated with VS-4718 alone in vivo revealed a discernible reduction in adhesion in the intact bone marrow niche of Prrx1-Cre; LSL-tdTomato recipient mice. VS-4718 treated leukemic cells localized to Prrx1-expressing perivascular endothelial cells and exhibited round morphology in contrast to the typical spindle-like appearance of Ikzf1-altered pre-B cells adhering to the bone marrow stroma, suggesting that VS-4718 treatment abolished the aberrant leukemic cell-stromal adhesion induced by Ikaros alterations in vivo. Conclusions: Direct inhibition of FAK with VS-4718 attenuates the adhesive, stem-like properties of IKZF1-altered BCR-ABL1 leukemic cells that contribute to the poor prognosis of patients treated with currently available therapies. Targeted FAK inhibition is thus a promising avenue for improving the response of BCR-ABL1 ALL to dasatinib, particularly in refractory cases harboring IKZF1 alterations. These data support the clinical development of VS-4718 in combination with dasatinib in Ph+ B-ALL. Disclosures Shapiro: Verastem: Employment, Equity Ownership. Pachter:Verastem: Employment, Equity Ownership. Weaver:Verastem: Employment, Equity Ownership. Mullighan:Amgen: Honoraria, Speakers Bureau; Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Research Funding; Incyte: Consultancy, Honoraria. Off Label Use: The FAK inhibitor VS-4718 for the treatment of BCR-ABL1 acute lymphoblastic leukemia in preclinical models.

Download Full-text

Origins of “late” relapse in childhood acute lymphoblastic leukemia with TEL-AML1 fusion genes

Blood ◽

10.1182/blood.v98.3.558 ◽

2001 ◽

Vol 98 (3) ◽

pp. 558-564 ◽

Cited By ~ 89

Author(s):

Anthony M. Ford ◽

Karin Fasching ◽

E. Renate Panzer-Grümayer ◽

Margit Koenig ◽

Oskar A. Haas ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Fusion Gene ◽

Lymphoblastic Leukemia ◽

Cell Receptor ◽

Good Prognosis ◽

Leukemic Cells ◽

Late Relapse ◽

Childhood All ◽

Medium Risk ◽

Aml1 Gene

Abstract Approximately 20% of childhood B-precursor acute lymphoblastic leukemia (ALL) has a TEL-AML1 fusion gene, often in association with deletions of the nonrearranged TEL allele.TEL-AML1 gene fusion appears to be an initiating event and usually occurs before birth, in utero. This subgroup of ALL generally presents with low- or medium-risk features and overall has a very good prognosis. Some patients, however, do have relapses late or after the cessation of treatment, at least on some therapeutic protocols. They usually achieve sustained second remissions. Posttreatment relapses, or even very late relapses (5-20 years after diagnosis), in childhood ALL are clonally related to the leukemic cells at diagnosis (by IGH or T-cell receptor [TCR] gene sequencing) and are considered, therefore, to represent a slow re-emergence or escape of the initial clone seen at diagnosis. Microsatellite markers and fluorescence in situ hybridization identified deletions of the unrearranged TEL allele and IGH/TCR gene rearrangements were analyzed; the results show that posttreatment relapse cells in 2 patients with TEL-AML1–positive ALL were not derived from the dominant clone present at diagnosis but were from a sibling clone. In contrast, a patient who had a relapse while on treatment with TEL-AML1 fusion had essentially the sameTEL deletion, though with evidence for microsatellite instability 5′ of TEL gene deletion at diagnosis, leading to extended 5′ deletion at relapse. It is speculated that, in some patients, combination chemotherapy for childhood ALL may fail to eliminate a fetal preleukemic clone with TEL-AML1 and that a second, independent transformation event within this clone after treatment gives rise to a new leukemia masquerading as relapse.

Download Full-text

Physiologic IGFBP7 levels prolong IGF1R activation in acute lymphoblastic leukemia

Blood Advances ◽

10.1182/bloodadvances.2020003627 ◽

2021 ◽

Vol 5 (18) ◽

pp. 3633-3646

Author(s):

Leonardo Luís Artico ◽

Angelo Brunelli Albertoni Laranjeira ◽

Livia Weijenborg Campos ◽

Juliana Ronchi Corrêa ◽

Priscila Pini Zenatti ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Insulin Receptor ◽

Lymphoblastic Leukemia ◽

Cell Types ◽

Therapeutic Interventions ◽

Igf Binding Proteins ◽

Childhood All ◽

Erk Phosphorylation

Abstract Insulin and insulin-like growth factors (IGFs) are mitogenic and prosurvival factors to many different cell types, including acute lymphoblastic leukemia (ALL). Circulating IGFs are bound by IGF binding proteins (IGFBPs) that regulate their action. IGFBP7 is an IGFBP-related protein (IGFBP-rP) that in contrast to other IGFBPs/IGFBP-rPs features higher affinity for insulin than IGFs and was shown to bind the IGF1 receptor (IGF1R) as well. The role of IGFBP7 in cancer is controversial: on some tumors, it functions as an oncogene, whereas in others, it functions as a tumor suppressor. In childhood ALL, higher IGFBP7 expression levels were associated with worse prognosis. Here we show that IGFBP7 exerts mitogenic and prosurvival autocrine effects on ALL cells that were dependent on insulin/IGF. IGFBP7 knockdown or antibody-mediated neutralization resulted in significant attenuation of ALL cell viability in vitro and leukemia progression in vivo. IGFBP7 was shown to prolong the surface retention of the IGF1R under insulin/IGF1 stimulation, resulting in sustained IGF1R, insulin receptor substrate 1 (IRS-1), protein kinase B (AKT), and extracellular signal-regulated kinase (ERK) phosphorylation. Conversely, the insulin receptor was readily internalized and dephosphorylated on insulin stimulation, despite IGFBP7 addition. The affinity of homodimeric IGF1R for insulin is reportedly >100 times lower than for IGF1. In the presence of IGFBP7, however, 25 ng/mL insulin resulted in IGF1R activation levels equivalent to that of 5 ng/mL IGF1. In conclusion, IGFBP7 plays an oncogenic role in ALL by promoting the perdurance of IGF1R at the cell surface, prolonging insulin/IGF stimulation. Preclinical data demonstrate that IGFBP7 is a valid target for antibody-based therapeutic interventions in ALL.

Download Full-text

Presence of Preleukemic Clones at Birth in the Majority of Children with B-Lineage Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v106.11.88.88 ◽

2005 ◽

Vol 106 (11) ◽

pp. 88-88

Author(s):

Bernd Gruhn ◽

Nadine Pfaffendorf ◽

Susan Wittig ◽

Roland Zell ◽

Ralf Häfer ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Mononuclear Cells ◽

Germ Line ◽

Fusion Gene ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Guthrie Card ◽

Childhood All ◽

Blood Spots ◽

B Lineage

Abstract The proof for the prenatal origin of childhood acute lymphoblastic leukemia (ALL) comes from the detection of concordant leukemia in monozygotic twins and the identification of translocation breakpoint genomic sequences at birth in a limited number of ALL patients with t(4;11) or t(12;21) chromosomal translocation. However, most patients with childhood ALL lack leukemia-specific fusion gene sequences. Therefore, we have used the rearranged immunoglobulin heavy chain (IgH) genes as a marker for the detection of preleukemic clones at birth. Guthrie card blood spots of 32 children with B-lineage ALL treated at our institution were available for this retrospective study. The ALL patients had a median age of 5 years (range, 15 months to 14 years) and had median presenting white blood cell (WBC) counts of 10150/μl (range, 800 to 103800/μl). In all patients a monoclonal IgH gene rearrangement was obtained from diagnostic bone marrow and sequenced. Clone-specific primers were designed using the specific D-N-J and N-D-N sequences. A two-stage polymerase chain reaction (PCR) using a semi-nested approach was developed to improve sensitivity and specificity of amplification. In all 32 patients, one leukemic cell could be detected in a background of 105 normal blood mononuclear cells. Nineteen of the 32 patients (59%) had detectable IgH gene rearrangements at birth using the sensitive semi-nested PCR. Sequencing of the PCR products obtained from Guthrie card blood spots revealed the identical sequences identified from diagnostic leukemic cells. The fetal characteristics of the leukemic cells were indicated by the small numbers of nucleotides inserted into the N region and the shortened D germ line segments. Interestingly, five of the six children (83%) with hyperdiploid ALL had detectable preleukemic clones at birth. Four of the five children (80%) with pro-B ALL, 13 of the 21 children (62%) with cALL and only two of the six children (33%) with pre-B ALL had preleukemic clones on their cards. We did not observe any differences in age at diagnosis or presenting WBC count between the 19 patients with preleukemic clones at birth and the 13 patients whose Guthrie cards were tested negative. Our results suggest that the majority of children with B-lineage ALL has preleukemic clones already at birth indicating a prenatal origin of leukemia. In addition, postnatal factors are important in leukemogenesis as well because of the long latency periods until clinical diagnosis of leukemia.

Download Full-text

MicroRNA Mir-125b Causes Leukemia

Blood ◽

10.1182/blood.v116.21.3158.3158 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3158-3158

Author(s):

Marina Bousquet ◽

Marian Harris ◽

Beiyan Zhou ◽

Mark D. Fleming ◽

Harvey Lodish

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Fetal Liver ◽

Lymphoblastic Leukemia ◽

Macrocytic Anemia ◽

Leukemic Cells ◽

Cell Acute Lymphoblastic Leukemia

Abstract Abstract 3158 MicroRNA miR-125b has been shown to be involved in different kind of leukemia. Indeed, the chromosomal translocation t(2;11)(p21;q23) found in patients with myelodysplasia and acute myeloid leukemia leads to an overexpression of miR-125b up to 90 fold. Moreover, miR-125b is also upregulated in patients with B-cell acute lymphoblastic leukemia carrying the t(11;14)(q24;q32) translocation. To decipher the presumed oncogenic mechanism of miR-125b, we used transplantation experiments in mice. All of the mice transplanted with fetal liver cells ectopically expressing miR-125b showed an increase in white blood cell count, in particular in neutrophils and monocytes, associated with a macrocytic anemia. Among these mice, half of them died of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, or a myeloproliferative disorder, suggesting an important role of miR-125b in myeloid and lymphoid lineages. Co-expression of miR-125b and the BCR-ABL fusion gene in transplanted cells accelerated the development of leukemia in mice, compared to control mice expressing only BCR-ABL, suggesting that miR-125b confers a proliferative advantage to the leukemic cells. Thus we showed that the overexpression of miR-125b is sufficient to induce leukemia in vivo and decrease the latency of BCR-ABL -induced leukemia. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Preclinical Activity of LBH589 Alone or in Combination with Chemotherapy in a Xenogeneic Mouse Model of Human Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v118.21.1520.1520 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1520-1520

Author(s):

Xabier Agirre ◽

Amaia Vilas-Zornoza ◽

Gloria Abizanda ◽

Cristina Moreno ◽

Victor Segura ◽

...

Keyword(s):

Cell Proliferation ◽

Acute Lymphoblastic Leukemia ◽

Mouse Model ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Regulatory Function ◽

Human Leukemia

Abstract Abstract 1520 Histone deacetylases (HDACs) have been identified as therapeutic targets due to their regulatory function in chromatin structure and organization. Here we analyzed the therapeutic effect of LBH589 or panobinostat, a class I-II HDAC inhibitor, in acute lymphoblastic leukemia (ALL). In vitro, LBH589 induced a significant dose-dependent increase in cell apoptosis and a markedly inhibition of cell proliferation, which were associated with increased H3 and H4 histone acetylation. While apoptosis of ALL cells was detected between 12 and 24 hours after treatment with LBH589, changes in acetylated H3 and H4 were detected as early as 2 hours. Phosphorylation of H2AX, as an early marker of DNA damaged, was detected 12 to 24 hours after in vitro treatment with LBH589. These results suggest that H3 and H4 acetylation precede DNA damaged and induction of apoptosis indicating that inhibition of HDAC is likely to be responsible at least in part for LBH589 induced apoptosis and inhibition of cell proliferation. The in vivo activity of LBH589 was initially examined in a subcutaneous ALL mouse model. The ALL cell lines TOM-1 and MOLT-4 were transplanted (1×106 cell per animal) subcutaneously into the left flanks of 6-week-old female BALB/cA-Rag2−/−γc−/−. These cell lines develop into a rapidly growing tumor. Treatment with 5mg/kg of LBH589 was initiated 24 hours after injection of the leukemic cells, included 3 cycles of 5 consecutive days of LBH589 with two days rest between cycles and animals were monitored for 24 days. A significant inhibition of tumor growth was demonstrated in animals treated with LBH589 compared with control animals (P <0.01). Inhibition of leukemia cell growth was associated with an increase in the levels of acetylated H3 and H4 and an increase in phosphorylated H2AX in the leukemic cells obtained after sacrifice of mice. These results suggest that LBH589 has a powerful antileukemic effect not only in vitro but also in vivo. Using primary ALL cells, a xenograft model of human leukemia in BALB/c-RAG2−/−γc−/− mice was established, allowing continuous passages of transplanted cells to several mouse generations. A total of 10 million cells from a patient with T-ALL (ALL-T1) and a patient with B-ALL (ALL-B1) were administered intravenously into the tail vein of 6-week-old immunodeficient female BALB/cA-Rag2−/−γc−/− mice. Kinetics of engraftment of leukemic cells was monitored in PB and BM by phenotyping while organ infiltration was analyzed by immunohistochemistry. There were no significant differences in the genome, methylome or transcriptome between the original sample and the samples obtained after multiple generations on mice. To determine the efficacy of LBH589 alone or in combination with drugs currently used for treatment of ALL, BALB/cA-RAG2−/−γc−/− mice engrafted with ALL-T1 and ALL-B1 cells were treated with LBH589, Vincristine and Dexamethasone or a combination of LBH589 with Vincristine and Dexamethasone. Treatment was initiated when disease could be detected in PB by FACS (24 hours after injection of cells for ALL-T1 and between day 17 and 21 after injection for ALL-B1). LBH589 was administered i.p. on days 1–5, 8–12 and 15–19, Vincristine i.v. on days 1, 8 and 21 and Dexamethasone daily until day 21 i.p. and survival was analyzed. Treatment of mice engrafted with T or B-ALL cells with LBH589 induced an in vivo increase in the acetylation of H3 and H4, which was accompanied with prolonged survival of LBH589-treated mice in comparison with those receiving Vincristine and Dexametasone. Notably, the therapeutic efficacy of LBH589 was significantly enhanced in combination with Vincristine and Dexametasone. Our results demonstrate the therapeutic activity of LBH589 in combination with standard chemotherapy in pre-clinical models of ALL and suggest that this combination may be of clinical value in the treatment of patients with ALL. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Characterization of childhood acute lymphoblastic leukemia xenograft models for the preclinical evaluation of new therapies

Blood ◽

10.1182/blood-2003-08-2911 ◽

2004 ◽

Vol 103 (10) ◽

pp. 3905-3914 ◽

Cited By ~ 178

Author(s):

Natalia L. M. Liem ◽

Rachael A. Papa ◽

Christopher G. Milross ◽

Michael A. Schmid ◽

Mayamin Tajbakhsh ◽

...

Keyword(s):

Patient Outcome ◽

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Receptor Gene ◽

Clonal Variation ◽

Clinical Relapse ◽

Childhood All ◽

Genetic Characteristics

Abstract Continuous xenografts from 10 children with acute lymphoblastic leukemia (ALL) were established in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Relative to primary engrafted cells, negligible changes in growth rates and immunophenotype were observed at second and third passage. Analysis of clonal antigen receptor gene rearrangements in 2 xenografts from patients at diagnosis showed that the pattern of clonal variation observed following tertiary transplantation in mice exactly reflected that in bone marrow samples at the time of clinical relapse. Patients experienced diverse treatment outcomes, including 5 who died of disease (median, 13 months; range, 11-76 months, from date of diagnosis), and 5 who remain alive (median, 103 months; range, 56-131 months, following diagnosis). When stratified according to patient outcome, the in vivo sensitivity of xenografts to vincristine and dexamethasone, but not methotrexate, differed significantly (P = .028, P = .029, and P = .56, respectively). The in vitro sensitivity of xenografts to dexamethasone, but not vincristine, correlated significantly with in vivo responses and patient outcome. This study shows, for the first time, that the biologic and genetic characteristics, and patterns of chemosensitivity, of childhood ALL xenografts accurately reflect the clinical disease. As such, they provide powerful experimental models to prioritize new therapeutic strategies for future clinical trials.

Download Full-text