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.

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.

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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.

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Bone marrow from children in relapse with pre-B acute lymphoblastic leukemia proliferates and disseminates rapidly in scid mice

Blood ◽

10.1182/blood.v78.11.2973.bloodjournal78112973 ◽

1991 ◽

Vol 78 (11) ◽

pp. 2973-2981 ◽

Cited By ~ 2

Author(s):

S Kamel-Reid ◽

M Letarte ◽

M Doedens ◽

A Greaves ◽

B Murdoch ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Poor Prognosis ◽

Lymphoblastic Leukemia ◽

Growth Characteristics ◽

Scid Mice ◽

Leukemic Cells ◽

Murine Bone Marrow ◽

In Vivo Growth

Bone marrow samples from patients with pre-B acute lymphoblastic leukemia (pre-B ALL), either at diagnosis or at relapse, were transplanted into scid mice to determine whether these freshly obtained leukemic cells could proliferate in vivo and whether there were any differences in their in vivo growth characteristics. Cells from three patients who relapsed within 13 months of diagnosis proliferated rapidly in the murine bone marrow, spleen, and thymus, invaded peripheral organs, and resulted in morbidity and mortality of the animals within 4 to 16 weeks. Cells from two patients who relapsed 3.5 years after diagnosis grew much slower than the early relapse samples, taking up to 30 weeks to infiltrate the bone marrow of recipient mice. In contrast, leukemic cells were absent or were detected at low numbers in scid mice transplanted with cells obtained at diagnosis from three patients who have not yet relapsed. These results show an increased ability of leukemic cells from patients with aggressive lymphoblastic leukemia of poor prognosis to proliferate in scid mice.

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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.

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Establishment of a Novel CNS Infiltrated Xenograft Model through Engraftment of Patient-Derived Acute Lymphoblastic Leukemic Cells Into NOD/SCID/γc Null Mouse

Blood ◽

10.1182/blood.v116.21.3248.3248 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3248-3248

Author(s):

Itaru Kato ◽

Akira Niwa ◽

Megumu Saito ◽

Hisanori Fujino ◽

Satoshi Saida ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Hematologic Malignancy ◽

Lymphoblastic Leukemia ◽

Xenograft Model ◽

Leukemic Cells ◽

Null Mouse ◽

Immunodeficient Mice ◽

Mouse Xenograft Model ◽

Nog Mice

Abstract Abstract 3248 Background and Purpose: Acute lymphoblastic leukemia (ALL) is the most common type of childhood hematologic malignancy. Although improvements in treatment regimen have raised the 5-year survival rate as high as 80% for pediatric ALL patients, a minority of patients with various risk factors, including central nervous system (CNS) infiltration continue to have poor prognosis. Recently, bone marrow (BM) microenvironments which support leukemic stem cells have become noticed as an important element which can influence treatment response and relapse of the disease. Although leukemic cells appear to be completely eradicated through treatment, they are thought to survive within bone marrow and/or extramedullary microenvironments, such as CNS, causing disease recurrence. However, little is known about the CNS microenvironment for leukemic cells because of the lack of appropriate animal model. Even though several investigators have tried to establish a CNS infiltrated model of leukemia, major limitation with these studies are the use of leukemic cell lines and the preconditioning of recipient mice, which did not represent CNS leukemia observed in patients. Here we report the establishment of a novel xenograft model for primary human ALL using NOD/SCID/γc null (NOG) mouse. Without irradiation, this model recapitulates CNS as well as extramedullary leukemic infiltration (hereby referred to as the h-leukemic NOG model). Result: Primary bone marrow samples were collected from 9 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 NOG mice. Primary samples from 8 out of 9 patients were successfully engrafted. Engrafted leukemic cells could be serially transplanted into secondary, tertiary and quaternary recipients. Morphological and FACS analyses revealed as high as 95% BM chimerism and showed that blast phenotypes were conserved through serial transplantations. Of note, extramedullary organs including the CNS, liver, spleen, and kidneys showed the leukemic invasion consistent with those of the donor ALL patients. Liver pathology in the h-leukemic NOG model is identical to that seen in the ALL patients. We also showed the existence of a functional niche in the liver mediated by SDF-1/CXCR4 axis. In terms of the CNS involvement, we observed the progressive infiltration of leukemic cells into the Virchow-Robin space that is consistent with the pathology of human ALL patients. Using this model, we examined the mechanism of dissemination and harboring of leukemic cells in the CNS niche. Discussion: NOG mice model for engraftment of human leukemic cells provides useful insights into the biology of ALL and allows us to answer various questions concerning the mechanism of extramedullary invasion and expansion. We have reported that NOG mice have significantly better human hematopoietic cell engraftment in the BM and extramedullary organs than other immunodeficient mice (Hiramatsu H. Blood. 2003), and is capable of supporting the growth of human neoplastic cells (Kato M. Nature. 2009). Here we report that this non-preconditioned mouse xenograft model reproduces leukemic extramedullary involvement, including the CNS, in sustaining leukemic cells. This approach provides a more sophisticated and physiological model suitable for the evaluation of molecular interactions between patient leukemic cells and host niche. Our h-leukemic NOG model will provide a powerful tool to analyze the CNS niche that harbors leukemia initiating cells. Moreover, this model would be a useful platform for developing novel anti-leukemic therapies that target CNS extramedullary niche. Disclosures: No relevant conflicts of interest to declare.

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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.

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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.

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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.

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Activated Fibroblasts Protect Acute Myeloid Leukemia Cells from Chemotherapy

Blood ◽

10.1182/blood.v124.21.5144.5144 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5144-5144

Author(s):

Yuanmei Zhai ◽

Jun Shi ◽

Yun Wan

Keyword(s):

Bone Marrow ◽

Myeloid Leukemia ◽

Poor Prognosis ◽

Leukemic Cells ◽

Leukemia Cells ◽

Protective Effects ◽

Bone Marrow Biopsies ◽

Activated Fibroblasts ◽

Acute Myeloid

Abstract Introduction: It has become clear that cancer is not only just a disease of the genes, but the tumor microenvironment (TME) plays an important role in cancer progression (Borriello L & DeClerck YA, 2014). In leukemia, the bone marrow plays a special role in environment-mediated drug resistance as it is not only a sanctuary protecting tumor cells from cytotoxic drugs, but also a source of many stromal cells that colonize primary tumors and contribute to the pre-metastatic niche (Borriello L & DeClerck YA, 2014; Meads MB, et al, 2008). As we all know, there exists two distinct BM niches in this significant microenvironment: ‘osteoblastic (endosteal)’ and ‘vascular’ niches (Tabe Y & Konopleva M, 2014; Konopleva M, et al, 2009). Besides these two, scholars transferred to focus on the stromal in bone marrow，which may have importance effects on leukemic cells (Wu S, et al, 2005; Campana D, et al, 2005). Shriram V. Nath et al. found that there was a negative correlation between reticulin fiber density (RFD) at diagnosis in childhood ALL and white blood cell count in peripheral blood. Similarly, there was a negative correlation between RFD and the percentage of blast cells in blood. They speculated fiber in the bone marrow leading to poor prognosis in acute lymphoblastic leukemia (ALL) via anchoring leukemia cells in bone marrow stromal (Nath SV, et al. 2011). But the role of fibers and its mainly producing cells fibroblasts has not been previously studied in acute myeloid leukemia (AML). Therefore, we retrospectively investigate the bone marrow biopsies of primary AML patients, to analyze the relationship between the RFD and prognosis in adult AML, and to identify and quantitative analyze protective effects of the fibroblasts on leukemia cells from chemotherapy in vitro. Methods: Makers of activated fibroblasts were stained by immunohistochemistry on bone marrow biopsies; The RFD were evaluated based on the grid point method of computer; Obtained activated fibroblasts by induced BM-MSC of primary AML with human recombinant TGFβ1.(4) Analyzed protective effects of activated fibroblasts by co-cultured with leukemia cells such as THP-1/K562, futher explored the mechanism via SB431542, a specific inhibitor of TGFβ signaling pathway. Result: RFD in primary AML was significant higher than control. Patients with higher RFD indicated poorer prognosis; However, collagenI coated plates showed no effects on survival rate of the leukemic cells. Here, we demonstrated that the general makers of activated fibroblasts: FSP1, α-SMA and FAP in primary AML were significant higher than control, implied that there existed amount of activated fibroblasts in patients; Activated fibroblast protect both THP-1 and K562 from apoptosis with treatment of Ara-c, and arrested them in phase of G0/G1. Moreover, this protect effects of activated fibroblast will be cancelled with SB431542. Conclusion: Activated fibroblasts and fibrous tissue were both proliferated abnormally in primary AML, and the later was significantly associated with relapse-free survival and overall survival of patients, indicating that RFD could be used as an important factor of poor prognosis and should guide clinical intervention. In vitro induced activated fibroblasts could be able to protect leukemia cells from chemotherapy. The possible mechanisms might be that the soluble cytokines TGFβ1, but not collagen I, which were secreted by activated fibroblasts, contributed to leukemia protection. In addition to this, activated fibroblasts could also change cell cycle of tumor cells, make them more arrested in G0/G1 phase, thus decreased the their chemotherapy sensitivity. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v128.22.278.278 ◽

2016 ◽

Vol 128 (22) ◽

pp. 278-278

Author(s):

Kathryn G. Roberts ◽

Olga Bridges ◽

Laura J. Janke ◽

Kevin Ebata ◽

Brian B Tuch ◽

...

Keyword(s):

Bone Marrow ◽

Lymphoblastic Leukemia ◽

Genetically Engineered ◽

Leukemic Cells ◽

Spleen Weight ◽

Constitutive Activation ◽

Pdx Model ◽

Genetically Engineered Mouse Model ◽

Bone Marrow Blasts

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.

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