Genetic Modeling and Therapeutic Targeting of ETV6-NTRK3 with Loxo-101in Acute Lymphoblastic Leukemia

Abstract Introduction: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype characterized by kinase-activating alterations. One recurrent alteration is the ETV6-NTRK3 fusion, which results in constitutive activation of NTRK3, a member of the neurotrophic receptor kinase family. ETV6-NTRK3 has been identified in a range of malignancies, including breast cancer, pediatric glioma and infantile fibrosarcoma. The oncogenic role of ETV6-NTRK3 in B-cell ALL has not been investigated. The goals of this study were to assess the development of leukemia in genetically engineered models of ETV6-NTRK3, and to investigate efficacy of the specific TRK A, B and C inhibitor, LOXO-101, currently in clinical trials for the treatment of solid tumor patients who harbor NTRK fusions. Methods: For in vitro studies, kinase fusions were expressed in IL3 dependent Ba/F3 cells. To generate a genetically engineered mouse model, we used a previously reported conditional knockin model of Etv6-NTRK3 (Cancer Cell 2007;12:542-558), whereby the human portion of NTRK3 cDNA encoding the tyrosine kinase domain was inserted into exon 6 of the mouse Etv6 locus, downstream of a floxed transcriptional terminator sequence. Expression of the Etv6-NTRK3 protein was accomplished using Cre-recombinase driven by the B-lineage promoter CD19. A patient derived xenograft (PDX) model of ETV6-NTRK3 was established by engrafting primary human ALL cells expressing luciferase into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Phosphoflow cytometry analysis and sensitivity to LOXO-101 was assessed in vitro and in vivo. Results: Etv6-NTRK3/+, CD19-Cre mice developed aggressive disease with 100% penetrance and a median latency of 38 days (n=27). The average body weight of Etv6-NTRK3/+, CD19-Cre mice was significantly reduced compared to age-matched Etv6-NTRK3/+ controls (13.9 vs 20.2g, p<0.001). We observed increased spleen weight in Etv6-NTRK3/+, CD19-Cre mice compared to controls (142 vs 71mg, p=0.02), but no difference in peripheral white blood counts (9.7 vs 13.4 x 109/L, p=0.3). Presence of the Etv6-NTRK3 fusion was confirmed in bone marrow samples by RT-PCR. Immunophenotyping of bone marrow indicated arrest at the pre-B stage (Hardy stage C: B220+, CD19+, CD43+, BP1+, IgM-), recapitulating human ALL. Pathological analysis using hematoxylin and eosin and B220 staining showed infiltration of leukemic cells into the bone marrow, spleen, liver and lung. Interestingly, we observed extensive infiltration of leukemic cells into the central nervous system, specifically ventral to the thoracic and lumbar vertebrae, and the meninges within the brain. Copy number alteration and sequence mutation analysis is currently being performed to determine additional genetic lesions. Leukemia cells from the bone marrow displayed constitutive activation of the MAPK pathway via pERK1/2. We next assessed the in vitro efficacy of the TRK inhibitors crizotinib, which also inhibits ALK, and a more specific inhibitor, LOXO-101. Compared to crizotinib (IC50 205 nM), LOXO-101 was 10 times more potent against BaF3-ETV6-NTRK3 cells (IC5017 nM), and had no effect on other kinase fusions (ABL1, ABL2, CSF1R, FLT3, JAK2) up to 10µM. In addition, LOXO-101 was remarkably selective for TRK A, B and C in a cytotoxicity screen of 77 human cancer cell lines as compared to crizotinib. Using a PDX model of ETV6-NTRK3, we demonstrate that treatment with LOXO-101 (200mg/kg/day p.o for six weeks) reduced leukemic infiltration to undetectable levels in the bone marrow (0 vs 75.8% human CD45/CD19 bone marrow blasts, n=5 each group) and spleen compared to vehicle-treated mice (splenic weight 316 vs 20mg, p<0.001). Notably, treatment with dexamethasone had a modest effect against this tumor (average 55.3% bone marrow blasts and spleen weight 134mg, n=5). Mice treated with LOXO-101 were still alive and leukemia-free four weeks after the cessation of treatment, as determined by Xenogen imaging. Conclusion: We have described the first genetically engineered mouse model of Ph-like ALL with an ETV6-NTRK3 fusion, and reported remarkable efficacy of LOXO-101 against the NTRK3 fusion, with complete suppression of leukemic cell proliferation when administered as a monotherapy. These findings warrant screening for ETV6-NTRK3 in newly diagnosed ALL patients, and testing the efficacy of LOXO-101 in combination with chemotherapy regimens. Disclosures Ebata: Loxo Oncology: Employment, Other: Shareholder. Tuch:Loxo Oncology: Employment, Other: Shareholder. Nanda:Loxo Oncology: Employment, Other: Shareholder. Mullighan:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Loxo Oncology: Research Funding.

Download Full-text

Colony formation in vitro by leukemic cells in acute lymphoblastic leukemia (ALL)

Blood ◽

10.1182/blood.v52.4.712.712 ◽

1978 ◽

Vol 52 (4) ◽

pp. 712-718 ◽

Cited By ~ 28

Author(s):

SD Smith ◽

EM Uyeki ◽

JT Lowman

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Bone Marrow Cells ◽

Lymphoblastic Leukemia ◽

Lymphoid Cells ◽

Assay System ◽

Leukemic Cells ◽

Specific Cell ◽

Specific Cell Surface

Abstract An assay system in vitro for the growth of malignant lymphoblastic colony-forming cells (CFC) was established. Growth of malignant myeloblastic CFC has been previously reported, but this is the first report of growth of malignant lymphoblastic CFC. Established assay systems in vitro have been very helpful in elucidating the control of growth and differentiation of both normal and malignant bone marrow cells. Lymphoblastic CFC were grown from the bone marrow aspirates of 20 children with acute lymphoblastic leukemia. Growth of these colonies was established on an agar assay system and maintained in the relative hypoxia (7% oxygen) of a Stulberg chamber. The criteria for malignancy of these colonies was based upon cellular cytochemical staining characteristics, the presence of specific cell surface markers, and the ability of these lymphoid cells to grow without the addition of a lymphoid mitogen. With this technique, specific nutritional requirements and drug sensitivities can be established in vitro, and these data may permit tailoring of individual antileukemic therapy.

Download Full-text

Serotherapy of acute lymphoblastic leukemia with monoclonal antibody

Blood ◽

10.1182/blood.v58.1.141.141 ◽

1981 ◽

Vol 58 (1) ◽

pp. 141-152 ◽

Cited By ~ 201

Author(s):

J Ritz ◽

JM Pesando ◽

SE Sallan ◽

LA Clavell ◽

J Notis-McConarty ◽

...

Keyword(s):

Monoclonal Antibody ◽

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Chemotherapeutic Agents ◽

Leukemic Cells ◽

Antigenic Modulation ◽

Multiple Intravenous Infusions ◽

Marrow Cellularity

Abstract We tested the efficacy of passive serotherapy in the treatment of acute lymphoblastic leukemia in four patients who had relapsed while receiving standard chemotherapeutic agents. Each patient received multiple intravenous infusions of J-5 monoclonal antibody specific for common acute lymphoblastic leukemia antigen (CALLA). In the three patients with circulating leukemic cells, there was a rapid decrease in circulating blasts that began immediately after antibody infusion, but not all leukemic cells were cleared, and remaining cells appeared to be resistant to further serotherapy. Although J-5 antibody was also demonstrable on bone marrow lymphoblasts immediately after antibody infusion in one patient, there was no change in bone marrow cellularity or differential during serotherapy. Analysis of the cell surface phenotype of leukemic cells during serotherapy and in vitro studies with patient cells suggests that resistance to serotherapy was mediated in part by antigenic modulation of CALLA in response to J-5 antibody.

Download Full-text

The Bone Marrow Niche of Patients with Acute Lymphoblastic Leukemia Produces No Increased Asparagine Levels In Vivo That May Lead to Clinical Asparaginase Resistance

Blood ◽

10.1182/blood.v118.21.1505.1505 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1505-1505

Author(s):

Wing H. Tong ◽

Rob Pieters ◽

Wim C.J. Hop ◽

Claudia Lanvers-Kaminsky ◽

Joachim Boos ◽

...

Keyword(s):

Bone Marrow ◽

Aspartic Acid ◽

Peripheral Blood ◽

Lymphoblastic Leukemia ◽

Mesenchymal Cells ◽

Leukemic Cells ◽

Significant Difference ◽

Trough Levels

Abstract Abstract 1505 Asparaginase is an essential component of combination chemotherapy of acute lymphoblastic leukemia (ALL). Asparaginase breaks down asparagine into aspartic acid and ammonia. Because asparagine is necessary for protein synthesis, its depletion leads to cell death. Recently, it has been suggested that mesenchymal cells in the bone marrow may produce asparagine and form ‘protective niches’ for leukemic cells. In vitro, this led to high levels of asparagine and asparaginase resistance of the ALL cells (Iwamoto et al. (J Clin Invest. 2007)). However, it is unknown if this holds true for the clinical in vivo situation. The aim of our study is to analyse whether mesenchymal cells or other cells in the bone marrow indeed produce significant amounts of asparagine in vivo that may lead to clinical asparaginase resistance. Ten de novo ALL patients were enrolled in this study. All children received induction chemotherapy according to protocol 1-A and 1-B of the Dutch Childhood Oncology Group (DCOG) ALL-10 protocol. Asparaginase levels and amino acid levels (asparagine, aspartic acid, glutamine and glutamic acid) were measured in bone marrow (BM) and peripheral blood at diagnosis (day 1), days 15, 33 and 79. On days that asparaginase was administered (days 15 and 33) it was ensured that study material was obtained before the E-coli L-asparaginase infusions. Changes over time of asparaginase trough levels in BM and peripheral blood were evaluated using Mixed models ANOVA. The amino acids levels in 0.5 ml BM, 3 ml BM and peripheral blood at days 15 and 33 were also compared using Mixed models ANOVA. All these analyses were done after log transformation of measured values to get approximate normal distributions. A two-sided p-value < 0.05 was considered statistically significant. The asparaginase levels were all below detection limit (< 5 IU/L) in BM and peripheral blood at days 1 and 79. In both compartments, the median asparaginase trough levels were not significantly different at days 15 and 33. At diagnosis, no significant difference in asparagine level between 3 ml BM and peripheral blood was found (median: 44.5 μM (range 20.6–59.6 μM) and 43.9 μM (range 18.4 –58.5 μM), respectively). However, the median level of aspartic acid at diagnosis in 3 ml BM (19.2 μM; range 6.2–52.6 μM) was significantly higher as compared to median level of peripheral blood (5.7 μM; range 2.4–10.1 μM) (p=0.002). The aspartic acid levels were also higher in BM compared to peripheral blood at days 15 and 33 (both p=0.001) and at day 79 (p=0.002). Aspartic acid levels were significantly higher in 0.5 ml versus 3 ml BM (p=0.001) and this difference was also found when comparing 0.5 ml BM versus peripheral blood (p<0.001) suggesting dilution with peripheral blood when taking higher volumes of ‘bone marrow’. Asparagine levels were all below the lower limit of quantification (LLQ < 0.2 μM) in both BM and blood during asparaginase treatment at days 15 and 33. At day 79, no significant difference in asparagine levels between BM (37.7 μM; range 33.4–50.3 μM) and peripheral blood (38.9 μM; range 25.7 –51.3 μM) was seen. During the time course of asparaginase infusions, the glutamine and glutamic acid levels did not change significantly. In conclusion, we demonstrate higher aspartic acid levels in bone marrow compared to peripheral blood. The higher aspartic acid levels are detected at diagnosis, during asparaginase therapy at days 15 and 33, and also at day 79 at complete remission, showing that these do not originate from leukemic cells nor from asparagine breakdown by asparaginase but from cells in the microenvironment of the bone marrow. However, there is no increased asparagine synthesis in vivo in the bone marrow of ALL patients. Therefore, increased asparagine synthesis by mesenchymal cells may be of relevance for resistance to asparaginase of leukemic cells in vitro but not in vivo. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽

10.1182/blood.v120.21.1506.1506 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1506-1506

Author(s):

Marika Masselli ◽

Serena Pillozzi ◽

Massimo D'Amico ◽

Luca Gasparoli ◽

Olivia Crociani ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Map Kinases ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Significant Proportion ◽

Leukemic Cells ◽

K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Colony formation in vitro by leukemic cells in acute lymphoblastic leukemia (ALL)

Blood ◽

10.1182/blood.v52.4.712.bloodjournal524712 ◽

1978 ◽

Vol 52 (4) ◽

pp. 712-718 ◽

Cited By ~ 1

Author(s):

SD Smith ◽

EM Uyeki ◽

JT Lowman

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Bone Marrow Cells ◽

Lymphoblastic Leukemia ◽

Lymphoid Cells ◽

Assay System ◽

Leukemic Cells ◽

Specific Cell ◽

Specific Cell Surface

An assay system in vitro for the growth of malignant lymphoblastic colony-forming cells (CFC) was established. Growth of malignant myeloblastic CFC has been previously reported, but this is the first report of growth of malignant lymphoblastic CFC. Established assay systems in vitro have been very helpful in elucidating the control of growth and differentiation of both normal and malignant bone marrow cells. Lymphoblastic CFC were grown from the bone marrow aspirates of 20 children with acute lymphoblastic leukemia. Growth of these colonies was established on an agar assay system and maintained in the relative hypoxia (7% oxygen) of a Stulberg chamber. The criteria for malignancy of these colonies was based upon cellular cytochemical staining characteristics, the presence of specific cell surface markers, and the ability of these lymphoid cells to grow without the addition of a lymphoid mitogen. With this technique, specific nutritional requirements and drug sensitivities can be established in vitro, and these data may permit tailoring of individual antileukemic therapy.

Download Full-text

Characteristics of Cell Proliferation in Children's Lymphoblastic Leukemia

Tumori Journal ◽

10.1177/030089166805400204 ◽

1968 ◽

Vol 54 (2) ◽

pp. 147-160 ◽

Cited By ~ 1

Author(s):

Alessandro Pileri ◽

Renzo Pietro Tarocco ◽

Felice Gavosto ◽

Alberto Ponzone ◽

Paolo Nicola

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Proliferative Activity ◽

Tritiated Thymidine ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Cell Diameter ◽

Acute Leukemias ◽

Peripheral Cells

The proliferative activity of the bone marrow and peripheral cells of acute infant lymphoblastic leukemia was evaluated by « in vitro » incorporation of thymidine-H3. The proliferative activity of leukemic lymphoblasts proved roughly similar to that already observed in hemocito-myeloblastic acute leukemias of adults. Within the lymphoblastic population, incorporation of tritiated thymidine was distributed very heterogeneously and there was no labelling of the smaller blasts. In the larger blasts, the labelling index increased progressively with the increase in cell diameter. The acute leukemia population can thus be divided into two classes: proliferating and non-proliferating. A study of the proliferative activity of lymphoblasts, contemporaneously in bone marrow and peripheral cells, suggested a division of cases into two groups. In one proliferative activity in the marrow was greater than in the peripheral blood; in the other it was equal to or less than in the peripheral blood. The second group was made up of cases whose clinical features presented a much more marked hepato-splenomegaly and high peripheral leucocytosis. A more detailed study of proliferative activity considering various classes of blasts within the same population showed that, in the first group of patients, the highest percentage of large blast cells is found at bone marrow level, while in the second the percentage of large blasts in the marrow is equal to or less than that observed in the peripheral blood. It was also shown that total proliferative activity is correlated to the percentage of large blasts. On the basis of these findings, one may admit that in first group forms the leukemia cells are generated prevalently in the bone marrow, while in second group forms most leukemic cells are formed elsewhere.

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

Blockage of SDF-1-CXCR4 Axis by AMD 3100 Can Be a Novel Therapy for Acute Lymphoblastic Leukemia by Targeting the Extramedullary Sites of Leukemic Cells.

Blood ◽

10.1182/blood.v114.22.981.981 ◽

2009 ◽

Vol 114 (22) ◽

pp. 981-981

Author(s):

Itaru Kato ◽

Akira Niwa ◽

Hisanori Fujino ◽

Katsutsugu Umeda ◽

Satoshi Saida ◽

...

Keyword(s):

Bone Marrow ◽

Survival Rate ◽

Poor Prognosis ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Normal Human ◽

Nog Mice ◽

Human Leukemic Cells ◽

Cxcr4 Axis

Abstract Abstract 981 Poster Board I-3 Background and Purpose: Acute lymphoblastic leukemia (ALL) is the most common type of childhood hematologic malignancy. Although the accumulated progresses in treatment regimen have raised the 5-year survival rate as high as 80% for whole pediatric patients, only poor prognosis, an overall survival rate of 30%, can be still now expected for the patients with relapsed diseases. Widespread extramedullary involvement such as liver, spleen, lymph nodes and central nervous system invasion is a well-known characteristic of ALL related to poor prognosis. Recently, bone marrow (BM) microenvironments supporting leukemic cells have been widely noticed as an important element which influences on treatment response and relapse of disease. Although the mechanism of extramedullary dissemination has been the most crucial issues in the study of leukemia, it still remains incompletely understood. In this study, we established a novel murine model of human ALL with NOD/SCID/γc null (NOG) mouse. Using this model, we examined the involvement of SDF-1-CXCR4 signaling axis in hepatomegary development in ALL. Result Primary bone marrow samples were collected from 13 children with ALL at the time of diagnosis with informed consent. The leukemic cells (1×106cells) were injected into the tail veins of non-irradiated 8- to 10-week old NOD/SCID/γc null (NOG) mice, a transgenic mouse with severe combined immunodeficiency and IL-2 receptor chain allelic mutation showing high potential to reconstitute the normal human hematopoietic system. Primary samples from 10 out of 13 patients were successfully engrafted into mice without any conditioning such as prior irradiation and DNA-damaging agents medication, and those engrafted leukemic cells were able to be serially transplanted into secondary, tertiary and quaternary recipients. Morphological and FACS analyses revealed as high as >80% blood chimerism and conserved blast phenotypes through serial transplantations. Moreover, extramedullary organs including liver, spleen and kidneys showed the leukemic invasion consistent with donor ALL disease. In contrast, no normal human hematopoiesis was observed in our xenotransplantation system without conditioning. CXCR4 is a known regulator of lymphocyte migration and has been suggested to be important for proliferation of normal leucocytes and leukemic cells. CXCR4 expression and function of leukemic cells in NOG mice were confirmed by flow cytometry and in vitro chemotaxis assays towards its known chemokine ligand SDF-1. Immunohistorical analysis of liver reveals that SDF-1 was detectable only in biliary duct endotherial cells. Finally, we demonstrated directly the effect of SDF-1-CXCR4 axis in our model by using the CXCR4 inhibitor AMD3100 in vivo and in vitro. Discussion: NOG mice model for engraftment of human leukemic cells provides significant insights into the biology of ALL and allows us to answer various questions concerning the molecular mechanism of extramedullaly invasion. This non-conditioning approach may prevent possible damage to the host microenvironment, thereby providing a more natural model for growth of human leukemic cells in mice. Our present study on the involvement of SDF-1-CXCR4 axis in ALL dissemination could rink to the novel therapies in future which target the extramedullary sites in order to perfectly overcome ALL. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Testing in Vitro Sensitivity of Minimal Residual Disease in Pediatric Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v120.21.2543.2543 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2543-2543

Author(s):

Line Groth-Pedersen ◽

Rebecca Valentin ◽

Kjeld Schmiegelow

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Cell Populations ◽

Sensitivity Testing ◽

Cell Death Assay ◽

In Vitro Sensitivity

Abstract Abstract 2543 Acute lymphoblastic leukemia (ALL) is the most frequent malignant disease in children and adolescents. Although improved risk grouping, anti-cancer treatment, and supportive care have resulted in survival rates above 80 %, 15–20 % of patients experience a relapse, which is associated with an inferior prognosis. Relapse is caused by persistence of minimal residual disease (MRD) primarily in the bone marrow during chemotherapy, and methods for identification of drugs that are capable of eliminating the MRD cell population is necessary, but not currently available. In vitro sensitivity testing has been carried out at the time of diagnosis and shows predictive value of treatment outcome and correlation to MRD levels. However, the in vitro sensitivity profiles have not been applicable for treatment stratification, since the vast majority of leukemic cells present at diagnosis are rather chemosensitive, as opposed to the MRD population. Thus, the more sensitive clones mask the more resistant ones. We hypothesized that in vitro sensitivity testing of the more resistant MRD cells remaining in bone marrow after the induction therapy could help to stratify patients to individualized chemotherapy. The greatest challenge in carrying out in vitro sensitivity testing on MRD cell populations is the low number of leukemic cells available, i.e. 0.1–5%. Traditionally, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flourometric microculture cytotoxicity assays have been used for in vitro sensitivity testing. They, however, need several million cells for in vitro sensitivity testing of 3 drugs. We aimed at developing a cell death assay applicable on small ALL cell populations. We show that a flow cytometry based assay using annexin V (reflecting exposure of phophatidylserine on the outer cell membrane during early apoptosis) and 7-Aminoactinomycin D (reflecting cell membrane rupture during late apoptosis) staining is a reliable method for evaluating early and late stages of cell death in small ALL cell populations. Both in ALL cell lines REH (B-cell precursor (BCP) ALL, t(12;21), Jurkat (t-ALL), RS4;11 (BCP ALL, t(4;11)) and Nalm-6 (BCP ALL, t(5;12)) and in primary ALL cell samples the results are highly reproducible and show the same relative sensitivity profile as when a large number of cells are used. This cell death assay only demands a total of 20.000 cells to determine the in vitro sensitivity for 3 drugs run in triplicates at 4 concentrations. This assay is thus applicable for sorted MRD populations accounting for 0.1% of the mononuclear cells in the bone marrow. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The NR4A Orphan Nuclear Receptors Do Not Confer Prednisolone resistance In Pediatric Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v122.21.3870.3870 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3870-3870

Author(s):

Ingrid M. Ariës ◽

Rosanna van den Dungen ◽

Rob Pieters ◽

Monique L. Den Boer

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Cell Survival ◽

Bone Marrow Cells ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Orphan Nuclear Receptors ◽

Qrt Pcr ◽

Phase Protein

Abstract Background Resistance against the glucocorticoid prednisolone still remains an obstacle for treatment of pediatric precursor B acute lymphoblastic leukemia (BCP-ALL) at initial diagnosis and even more after relapse. The molecular mechanisms behind prednisolone resistance in pediatric BCP-ALL is poorly defined. The NR4A family, consisting of NR4A1 (Nur77), NR4A2 (Nurr1) and NR4A3 (Nor1), are orphan nuclear receptors, which antagonize the glucocorticoid receptor. We hypothesized that upregulated NR4A family expression is responsible for prednisolone resistance in BCP-ALL. Methods Newly diagnosed pediatric acute lymphoblastic leukemia patients’ cells were isolated from bone marrow aspirates and only samples with ≥ 90% leukemic blasts were used in the present study. Gene expression microarrays of 178 BCP-ALL patients tested for in vitro prednisolone resistance were analyzed with Limma R Package in the statistical environment R, version 2.15.0. Microarray expression levels were confirmed using qRT-PCR. Nur77, Nurr1 and Nor1 protein expression in primary BCP-ALL patients’ were assessed with reverse phase protein array. Leukemic patients’ cells were transfected with labeled siRNA against NR4A1, NR4A2, and NR4A3, simultaneously, or with labeled siScrl, using the transfection reagent Dharmafect 4. Hereafter, cytotoxicity to prednisolone was determined by the in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) drug-resistance assay. Furthermore, viability of transfected cells was counted by trypan blue exclusion assay and cells were harvested after 72 hours of culture for RNA and protein isolation. Knockdown was confirmed with qRT-PCR and Western blot. Results In this study of 178 precursor BCP-ALL patients we discovered a 3.0-fold (p=0.007) raise in NR4A1, NR4A2, and NR4A3 microarray mRNA expression in in vitro prednisolone resistant compared to sensitive BCP-ALL patients’ cells, which was confirmed by qRT-PCR. In addition, reverse phase protein array identified a 2.7-fold (p<0.001) increased Nurr1 (NR4A1) and Nur77 (NR4A2) protein expression in in vitro prednisolone resistant compared to sensitive BCP-ALL patients’ cells. Nurr1 and Nur77 protein levels were overall 5-fold (p<0.001) higher in BCP-ALL patients compared to normal bone marrow cells. Simultaneous siRNA mediated knockdown of Nur77, Nurr1 and Nor1 in pediatric leukemic patients’ ALL cells decreased leukemic cell survival by 25.4±11.1% (p=0.029), but did not sensitize these cells to prednisolone (n=4). Conclusion The NR4A family members are higher expressed in prednisolone resistant ALL patients’ cells. Although literature describes an antagonizing effect of the NR4A family members on the glucocorticoid receptor, we could not find a functional contribution of the NR4A family to prednisolone resistance in BCP-ALL. We furthermore discovered an increased NR4A family expression in leukemic cells of BCP-ALL patients compared to normal bone marrow cells. Targeting of NR4A genes impaired cell survival. However, compensatory mechanisms exist and consequently all three NR4A members need to be targeted simultaneously to diminish cell survival. These data therefore suggest that the NR4A genes are not suitable to reverse prednisolone resistance nor to kill leukemic cells by targeted drugs. Disclosures: No relevant conflicts of interest to declare.

Download Full-text