Zeta-Chain-Associated Protein Kinase-70 (Zap-70) Promotes Acute Lymphoblastic Leukemia (ALL) Infiltration into the Central Nervous System By Enhancing Chemokine Receptor 7 (CCR7) Expression

Abstract Most children with B-cell precursor ALL (BCP-ALL) have an excellent prognosis but some patients are susceptible to CNS relapse, the mechanisms of which are largely unknown. Zap-70 is a tyrosine kinase mainly expressed in normal T- and NK-cells, but it is also expressed at low levels in normal B-cells. Zap-70 is however overexpressed in B-cell chronic lymphocytic leukemia (B-CLL) and is associated with an aggressive course of the disease. In B-CLL, Zap-70 was shown to up-regulate the expression of CCR7 (Calpe et al., 2011), which in turn allows T-lymphoblasts to enter the CNS (Buonamici et al., 2009). We hypothesized that Zap-70 mediated upregulation of CCR7 may enhance the homing and the survival of CNS-prone BCP-ALL cells in the CNS niche. We first transduced the 697 BCP-ALL cell line with a lentiviral vector carrying a non-targeting GFP (shGFP) or a Zap-70 (shZap-70) specific shRNA. Down-regulation of Zap-70 resulted in a significantly reduced CCR7 mRNA and surface protein expression. 697shGFP and 697shZap-70 cells were then injected intravenously into NSG mice, which were sacrificed when leukemic symptoms developed. There were no statistically significant differences in leukemic bone marrow infiltration and survival between the groups. However, immunohistochemical scoring of CNS infiltration (Krause et al., 2015) revealed that 9/10 animals (90%) in the 697shGFP group were CNS+ and only 2/7 animals (29%) in the 697shZap-70 group showed a CNS+ status (p = 0.0345). This suggests that Zap-70 mediated reduction in CCR7 impairs the CNS-positive phenotype of 697 cells in vivo. We next investigated if the Zap-70/CCR7 axis influences the ability of primary samples from patients to infiltrate the CNS of xenografts. Zap-70 mRNA was measured in pediatric BCP-ALL patients and patients with Zap-70 expression levels higher than the median were considered Zap-70high, and the remaining patients Zap-70low. 1 x 106 cells of 5 Zap-70high and 5 Zap-70low samples were injected intrafemorally into duplicate NSG mice. Interestingly, 7/10 (70%) of the mice injected with Zap-70high cells showed a CNS+ phenotype, whereas only 1/10 (10%) of the mice bearing Zap-70low samples were CNS+ (p = 0.0198). Importantly, leukemic cells of one Zap-70high patient purified from the xenograft CNS showed a marked upregulation of CCR7 expression as compared to cells isolated from bone marrow or spleen. These data suggest that the Zap-70/CCR7 axis is beneficial for patient BCP-ALL cells in the CNS of NSG mice. To test whether Zap-70/CCR7 is associated with CNS involvement in patients, we analyzed Zap-70 and CCR7 mRNA expression in diagnostic primary material of 76 BCP-ALL patients. The cohort contained 21 CNS-positive and 55 CNS-negative patients and was previously published by Cario et al. (2007). Patients were grouped into either Zap-70high/Zap-70low or CCR7high/CCR7low groups according to expression levels higher or lower than the median of the respective gene. Zap-70 expression was highly correlated with CCR7 expression (p = 0.0003; Spearman r = 0.401). There were no statistically significant differences in outcome surrogates such as prednisone response or MRD and there was no correlation between Zap-70/CCR7 expression and outcome (relapse or death). However, a trend towards high CCR7 levels and the occurrence of death could be observed (p = 0.055). Interestingly, high expression of Zap-70 or CCR7 correlated with the presence of blasts in the cytospin of the initial cerebrospinal fluid sample (p = 0.014 and p = 0.045, respectively). Furthermore, there was a significant correlation between high Zap-70 and a higher CNS status and a trend between high CCR7 and a higher CNS status according to AIEOP-BFM 2009 criteria (p = 0.024 and p = 0.098, respectively). We finally performed comparative correlations in 48/76 patients (63.2%) that were Zap-70high/CCR7high (n=24) or Zap-70low/CCR7low (n=24). Most importantly, a significant correlation between Zap-70high/CCR7high expression and a higher CNS status could be observed (p = 0.009). These data suggest that a high expression of Zap-70 or CCR7 is associated with CNS infiltration in BCP-ALL patients and that measurements of Zap-70 and CCR7 expression should be combined. Altogether, we show that Zap-70/CCR7 is important for BCP-ALL cells to enter the CNS and to survive in that niche. We propose Zap-70/CCR7 as a mechanism of CNS leukemia and a diagnostic marker with potential therapeutic implications. Disclosures No relevant conflicts of interest to declare.

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Lineage Switch Under Blinatumomab of a Relapsed Common ALL Co-Expressing Myeloid Markers without MLL Rearrangement

Blood ◽

10.1182/blood.v128.22.5196.5196 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5196-5196

Author(s):

Annabel Zoghbi ◽

Beate Winkler ◽

Udo zur Stadt ◽

Ingo Müller ◽

Gabriele Escherich

Keyword(s):

Bone Marrow ◽

T Cells ◽

B Cell ◽

Conflicts Of Interest ◽

Therapeutic Option ◽

Lymphoblastic Leukemia ◽

Palliative Therapy ◽

Bone Marrow Aspiration ◽

Leukemic Cells ◽

Lineage Switch

Abstract Lineage switch under Blinatumomab of a relapsed Common ALL co-expressing myeloid markers without MLL rearrangement Annabel Zoghbi, Udo zur Stadt, Beate Winkler, Ingo Müller and Gabriele Escherich Background The bispecific antibody blinatumomab induces T cell-mediated B cell lysis by adhering to CD3 positive cytotoxic T cells and attaching to CD19, a marker which is commonly seen in lymphoblastic malignancies of the B cell lineage. It has been proven to be an effective therapeutic option for adult and pediatric refractory or relapsed B cell precursor acute lymphoblastic leukemia (ALL). Lineage switch from ALL to a CD19 negative myeloid phenotype is well known in patients with mixed lineage leukemia (MLL) gene rearrangements and has been recently described following blinatumomab treatment in an infant with MLL rearrangement as well as for two patients with MLL rearrangement who were treated with CAR-T cells. Case description A nine year old girl with common ALL ( strong positivity for CD 10,19, 24,34,45, 52 and 79a) with the coexpression of myeloid markers (CD13 86% and CD66c 56%) was treated according to CoALL 08-09 high risk protocol and showed a very slow MRD response. Cytogenetic studies showed a hyperdiploid karyotyp without evidence of a MLL, BCR/ABL or TEL/AML rearrangement. Only one month into maintenance therapy she relapsed with the phenotype of the initial blasts. Because of non-response (M3 marrow) after the first two cycles of treatment accorting to the IntReALL protocol with a M3 marrow. The patient became aplastic for 2 months and developed aspergillus pneumonia, which was treated by combination of antifungals and granulocyte infusions. Nevertherless, blinatumomab treatment was initiated. The patient tolerated treatment well and did not develop a cytokine release syndrome, but eventually recovered granulopoiesis under blinatumomab treatment. Leukemic blasts below the 1% border, however, were detectable throughout immunotherapy. In the absence of other opportunities, we planned on a HSCT and in the event of relapse thereafter re-initiation of Blinatumomab in the allogeneic setting with the new immune system developing. Hence, the patient underwent an uneventful MUD-HSCT in March 2016 following conditioning with TBI/VP-16 and serotherapy with alemtuzumab due to CD52 expression of the blasts. On day 29 after transplant, a routine bone marrow aspiration revealed a second molecular relapse at the order of 2 x 10-3with the same immunophenotype. Thus, immunosuppression with cyclosporine A was drastically lowered and a grade II skin GvHD developed, which was controlled by topic treatment, before CsA was discontinued. Blinatumomab was reinitiated on day 49 after SCT. After two and a half weeks on therapy, an increase in LDH and leukocytes was visible. Bone marrow aspiration showed disease progression with 70% blasts, which now were CD19 negative with the immunophenotypical and morphological attributes of myeloid leukemia. Nevertheless, initial MRD markers still were strongly positive, indicating the common precursor with the initial phenotype. Detailed analysis will be presented. Cytogenetic analysis of the relapse samples revealed no changes. With no curative approach remaining, palliative therapy with cytarabine and thioguanine was initiated which could not control the fulminant expansion of leukemic cells, leading to the patient's death within four days from diagnosis. Conclusion CD19 negative relapses following blinatumomab therapy have been perceived in recent treatment studies, while reports on immunologic and morphologic presentation of a myeloid phenotype are scarce. Our patient coexpressed myeloid markers, CD13 and CD66c, from the start, thus, it is possible that subclones who did not carry CD19 had a pronounced selection advantage during blinatumomab treatment, leading to their vast expansion. Disclosures No relevant conflicts of interest to declare.

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Disturbed CXCR4/CXCL12 Axis In Pediatric Precursor B-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v122.21.2643.2643 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2643-2643 ◽

Cited By ~ 1

Author(s):

Lieke C.J. van den Berk ◽

Arian van der Veer ◽

Marieke E. Willemse ◽

Myrte J.G.A. Theeuwes ◽

Mirjam W. Luijendijk ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Serum Levels ◽

Initial Diagnosis ◽

Leukemic Cells ◽

Cxcr4 Receptor ◽

Cell Acute Lymphoblastic Leukemia

Abstract Malignant cells that infiltrate the bone marrow (BM) interfere with the normal cellular behavior of supporting cells, thereby creating an alternative malignant niche. This intercellular communication is mostly mediated by cytokines and their receptors. In this study, we find that expression of the CXCR4 receptor is significantly increased in pediatric precursor B-cell acute lymphoblastic leukemia (BCP-ALL) cells compared with normal mononuclear hematopoietic cells derived of the bone marrow (p=0.016). Furthermore, we show that high CXCR4 expression is correlated with an unfavorable clinical outcome in BCP-ALL (5-yr CIR ±SE: 38.4% ±6.9% in CXCR4-high versus 12.0% ±4.6% in CXCR4-low expressing patients, p<0.001). Interestingly, BM serum levels of the CXCR4 ligand (CXCL12) are 2.7-fold lower (p=0.005) in samples taken at initial diagnosis of BCP-ALL compared with the levels in samples taken of non-leukemic controls. We show that induction chemotherapy restores CXCL12 levels in the BM to normal levels. Blocking the CXCR4 receptor with Plerixafor (FDA-approved drug) showed that the lower CXCL12 serum levels at initial diagnosis could not be explained by consumption by the leukemic cells, nor did we observe an altered CXCL12-production capacity of BM-MSC at this time-point. We rather observed that a very high density of leukemic cells negatively affected CXCL12 production by the BM-MSC while stimulating the secretion levels of G-CSF. These results suggest that highly proliferative leukemic cells are able to down-regulate the production of cytokines involved in homing (CXCL12), while simultaneously up-regulating the production of cytokines involved in hematopoietic mobilization (G-CSF). This disbalance may stimulate the spreading of BCP-ALL outside the BM. The data presented here suggest that interference with the CXCR4/CXCL12 axis (for instance by using Plerixafor) may be an effective way to mobilize BCP-ALL cells; the more ALL cells become mobilized, the less ALL cells may escape from combination chemotherapy. In proof-of concept studies, this hypothesis needs to be validated to pave the way for implementation in future treatment protocols for children with ALL. Disclosures: No relevant conflicts of interest to declare.

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B-Cell Precursor Acute Lymphoblastic Leukemia Cells Create a Self-Reinforcing Niche Independent of the CXCR4/CXCL12 Axis

Blood ◽

10.1182/blood.v126.23.1297.1297 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1297-1297

Author(s):

Bob de Rooij ◽

Roel Polak ◽

Rob Pieters ◽

Monique L. Den Boer

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Hematopoietic Cells ◽

Leukemic Cells ◽

Cell Precursor ◽

Cxcr4 Antagonist

Abstract Background Acute lymphoblastic leukemia (ALL) cells create a leukemic niche that protects malignant cells from the effects of cytostatic agents and immune cells by altering their bone marrow microenvironment. This malignant process can be counteracted by impairing the homing of leukemic cells towards the bone marrow. Hematopoietic cells express the chemokine receptor CXCR4 and migrate towards its ligand CXCL12, which is actively produced by MSCs in the bone marrow. Therefore clinical trials have been initiated using the CXCR4 antagonist AMD3100 (Plerixafor) during leukemia treatment. However, these trials, as well as priming of AML in more than 4000 patients using a CXCR4 dependent mechanism, have not resulted in improved overall survival rates. This suggests that CXCR4 inhibition is not sufficient to disrupt leukemic niches. Objectives In this study we investigated how leukemic cells regulate the chemoattractive properties of their microenvironment. Results Here we show, using an ex vivo niche model with primary MSCs, that B-cell precursor ALL (BCP-ALL) cells affect their healthy microenvironment without altering CXCL12 secretion. Using a transwell migration assay we studied the chemoattractive properties and chemokine secretion patterns of several cell types and co-cultures. We confirmed that BCP-ALL cells migrate towards a CXCL12 gradient produced by primary MSCs (11-fold more migrated cells compared to background, p < 0.001). Inhibition of CXCR4 by AMD3100 reduced migration towards MSCs by 80% (p < 0.01). BCP-ALL cells migrated even more towards co-cultures of BCP-ALL cells and primary MSCs (24-fold more migrated cells compared to background, p < 0.001). Strikingly, this ex vivo leukemic niche did not produce higher levels of CXCL12 compared to MSC mono-cultures. Moreover, the induced migration towards MSC-ALL co-cultures could not be inhibited by AMD3100 treatment, indicating that BCP-ALL cells enhance the chemoattractive properties of their microenvironment in a CXCL12-independent manner. In contrast to BCP-ALL cells, the migration of CD34+ progenitor cells towards co-cultures of BCP-ALL cells and MSCs was significantly reduced (0.8-fold compared to migration towards MSCs, p < 0.05). Similar results were observed when we studied the migratory behavior of MSCs. MSCs actively migrated towards BCP-ALL cells (1.7 fold compared to background, p < 0.001), while migration of MSCs was significantly reduced towards MSC-ALL co-cultures (0.4-fold compared to migration towards BCP-ALL, p < 0.001). To find candidate factors influencing this process, we quantified the secreted levels of 64 cytokines in co-cultures of patient-derived BCP-ALL cells and MSCs. We observed leukemia-driven cytokine secretion patterns that were not influenced by the source of primary MSCs. In contrast to unaltered levels of CXCL12, we observed significant inductions of MCP-1/CCL2 and MDC/CCL22 (CCR4-ligands), IL8 and GRO-1 (CXCR1/2-ligands) and IP10/CXCL10 (CXCR3-ligands). Conclusion Our data indicate that leukemic cells alter the chemoattractive properties of their microenvironment, resulting in the secretion of multiple chemokines into the leukemic niche. This leukemic niche is highly potent in attracting BCP-ALL cells and repels the influx of healthy hematopoietic cells and MSCs using a CXCL12-independent mechanism. Furthermore, our results identify candidate factors that might be valuable future therapeutic targets. Disclosures No relevant conflicts of interest to declare.

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Anlotinib Shows Potent Antileukemia Effects in B-Cell Acute Lymphocytic Leukemia through the Blockage of Angiogenic Related Pathways

Blood ◽

10.1182/blood-2020-141039 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 49-49

Author(s):

Qiuling Chen ◽

Yuelong Jiang ◽

Qinwei Chen ◽

Long Liu ◽

Bing Xu

Keyword(s):

B Cell ◽

Solid Tumors ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Unmet Need ◽

Lymphocytic Leukemia ◽

Advanced Lung Cancer ◽

Antitumor Effects

Acute lymphoblastic leukemia (ALL) derives from the malignant transformation of lymphoid progenitor cells with ~85% being originated from B-cell progenitors (B-ALL). Despite fairly good prognoses for most pediatric B-ALL patients, the outcome is fatal in over 50% of adult patients who have a recurrent or progressive disease and lack of effective therapeutic approaches. Therefore, novel treatment strategies with high efficacy and low toxicity are an unmet need for B-ALL patients, especially those with relapsed or refractory status. Angiogenesis is a process of new vessel formation that requires the participation of multiple proangiogenic factors (e.g., VEGF, PDGF, and FGF) and their corresponding receptors (e.g., VEGFR, PDGFR, and FGFR). Angiogenesis, a well-established feature of solid tumors, also contributes to leukemia progression and correlates with the involvement of specific sanctuary sites in ALL, highlighting that the perturbation of angiogenesis would be an attractive approach for ALL treatment. Anlotinib is an oral tyrosine kinase (TKI) inhibitor with a broad range of antitumor effects via the suppression of VEGFR, PDGFR and FGFR. Of importance, anlotinib has been approved for the treatment of advanced lung cancer in China. Here, we evaluated the antileukemia activity of anlotinib in preclinical B-ALL models and its underlying molecular mechanisms. In this study, we observed that anlotinib significantly blunted the capability of cell proliferation and arrested cell cycle at G2 phase in B-ALL cell lines. Subsequently, we found that anlotinib resulted in remarkably enhanced apoptosis in B-ALL in vitro. To assess the in vivo antileukemia potential, we established a B-ALL patient-derived xenograft (PDX) mouse model and then treated the B-ALL PDX model with anlotinib. As a result, oral administration of anlotinib pronouncedly delayed in vivo B-ALL cell growth and reduced leukemia burden with acceptable safety profiles in this model. As for the mechanism of action, the antileukemia effect of anlotinib was associated with the disruption of the role of VEGFR2, PDGFRb, and FGFR3. Moreover, we revealed that this drug blocked the PI3K/AKT/mTOR/ signaling, a pathway that is linked with angiogenesis and its proangiogenic regulators, including VEGFR2, PDGFRb, and FGFR3. In aggregate, these results indicate that anlotinib is a potent antitumor agent for the treatment of B-ALL via the inhibition of angiogenic relevant pathways, which provide a novel potential treatment intervention for patients with B-ALL who have little effective therapy options. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Anlotinib originally designed by China is a novel orally active multitarget inhibitor that is evaluating in clinical trials against multiple solid tumors.

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The influence of a dosage regimen of dexamethasone on detection of normal B-cell precursors in the bone marrow of children with BCP-ALL at the end of induction therapy

Pediatric Hematology/Oncology and Immunopathology ◽

10.24287/1726-1708-2020-19-1-53-57 ◽

2020 ◽

Vol 19 (1) ◽

pp. 53-57

Author(s):

E. V. Mikhailova ◽

T. Yu. Verzhbitskaya ◽

J. V. Roumiantseva ◽

O. I. Illarionova ◽

A. A. Semchenkova ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cell ◽

Induction Therapy ◽

Therapy Group ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Intermittent Therapy ◽

Continuous Therapy ◽

B Cell Precursors

Minimal residual disease (MRD) monitoring by flow cytometry at the end of induction therapy is one of the key ways of a prognosis assessment in patients with acute lymphoblastic leukemia (ALL). In B-cell precursor ALL (BCP–ALL), this method of MRD detection is complicated due to the immunophenotypic similarity between leukemic cells and normal B-cell precursors (BCPs). A decrease in intensity of induction therapy can lead to a more frequent appearance of normal BCPs in the bone marrow, which significantly complicates the MRD monitoring. Aim: to assess the incidence of normal BCPs in bone marrow on the 36th day of induction therapy with two different regimens of glucocorticoid (GC) administration according to ALL-MB 2015 protocol. This study was approved by the Independent Ethical Committee and the Academic Council of Dmitriy Rogachev National Medical Research Center of Pediatric Hematology, Oncology, Immunology Ministry of Healthcare of Russian Federation. The study included 220 patients with BCP-ALL who were randomized to two types of GC-based induction therapy: a continuous administration of dexamethasone (n = 139) and an intermittent regimen with a 1-week dexamethasone therapy stop (n = 81). On the 36th day of induction therapy, MRD and normal BCPs were quantified in bone marrow samples by flow cytometry. On the 36th day of treatment, 43.2% of BCP(+) samples were established in the intermittent-therapy group, and 27.3% in the continuous-therapy group (p = 0.016). Comparison of the BCP level in BCP(+) samples revealed the more equitable distribution of BCPs at different developmental stages in the intermittent-therapy group, meanwhile mainly the immature BCPs in a quantity of less than 0.01% were found in the continuous-therapy group. Reduced-intensity induction therapy for patients with BCP-ALL leads to a noticeable increase of normal BCPs in bone marrow at the end of this treatment stage. A higher rate of BCP(+) bone marrow samples hinder the MRD detection due to the immunophenotypic similarity of BCPs and leukemic cells.

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Adoptive T-Cell Therapy for B-Cell Acute Lymphoblastic Leukemia: Preclinical Studies

Blood ◽

10.1182/blood.v94.10.3531.422k14_3531_3540 ◽

1999 ◽

Vol 94 (10) ◽

pp. 3531-3540 ◽

Cited By ~ 22

Author(s):

Angelo A. Cardoso ◽

J. Pedro Veiga ◽

Paolo Ghia ◽

Hernani M. Afonso ◽

W. Nicholas Haining ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

B Cell ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Adoptive T Cell Therapy ◽

Leukemic Cells ◽

Acute Leukemias ◽

T Cell Lines

We have previously shown that leukemia-specific cytotoxic T cells (CTL) can be generated from the bone marrow of most patients with B-cell precursor acute leukemias. If these antileukemia CTL are to be used for adoptive immunotherapy, they must have the capability to circulate, migrate through endothelium, home to the bone marrow, and, most importantly, lyse the leukemic cells in a leukemia-permissive bone marrow microenvironment. We demonstrate here that such antileukemia T-cell lines are overwhelmingly CD8+ and exhibit an activated phenotype. Using a transendothelial chemotaxis assay with human endothelial cells, we observed that these T cells can be recruited and transmigrate through vascular and bone marrow endothelium and that these transmigrated cells preserve their capacity to lyse leukemic cells. Additionally, these antileukemia T-cell lines are capable of adhering to autologous stromal cell layers. Finally, autologous antileukemia CTL specifically lyse leukemic cells even in the presence of autologous marrow stroma. Importantly, these antileukemia T-cell lines do not lyse autologous stromal cells. Thus, the capacity to generate anti–leukemia-specific T-cell lines coupled with the present findings that such cells can migrate, adhere, and function in the presence of the marrow microenvironment enable the development of clinical studies of adoptive transfer of antileukemia CTL for the treatment of ALL.

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Disease Propagating Blasts in Standard and High Risk Acute Lymphoblastic Leukemia Are Frequent and of Diverse Immunophenotype.

Blood ◽

10.1182/blood.v114.22.1421.1421 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1421-1421 ◽

Cited By ~ 1

Author(s):

Klaus Rehe ◽

Kerrie Wilson ◽

Hesta McNeill ◽

Martin Schrappe ◽

Julie Irving ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

High Risk ◽

Cancer Stem Cells ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Color Flow ◽

Nsg Mice ◽

Limiting Dilution

Abstract Abstract 1421 Poster Board I-444 Conflicting results in the field of cancer stem cells have reignited debate regarding the frequency and identity of cells with the ability to self renew and to propagate the complete phenotype of the malignancy. Initially it was suggested by different studies that cancer stem cells represent only a small minority of the malignant population and that the immunophenotypes of these cells resemble a rather immature type in the cell hierarchy. More recent data from our own and other groups have challenged these findings by demonstrating that cells at different maturity levels within the leukemic hierarchy have cancer stem cell abilities and that the frequency of the leukemia maintaining cell is higher than previously thought (Cancer Cell 2008, 14(1), p47-58). We use an in vivo NOD/scid IL2Rγnull (NSG) mouse intra-femoral transplant model to determine the clonogenicity of sorted candidate leukemic stem cell populations, characterized by specific immunophenotypes. We selected the surface markers CD10 and CD20, in order to differentiate between rather immature and more mature cells. Furthermore we carried out limiting dilution experiments on sorted (CD20) and unsorted leukemic blasts to investigate the frequency of the proposed leukemic stem cells. Flow sorted ALL blasts of CD19+CD20low and CD19+CD20high as well as of CD19+CD10low and CD19+CD10high immunophenotype were transplanted into NSG mice. Sorts were performed on primary patient material and on leukemic blasts that had been harvested following prior passage in mice. Different subtypes of ALL were included (high risk: BCR/ABL (t9;22) positive (patients L4967, L4951, L49101, L8849, L2510), high hyperdiploid/MRD positive high risk (L754, L835), intermediate risk: high WBC/MRD negative (L736, L784), age >10 years (L803)). CD20 sorts were performed on primary patient material (L4951, L49101, L754, L835 and L776), on secondary samples harvested from engrafted primary mice (L4967, L4951, L2510, L736 and L754) and on tertiary samples harvested from engrafted secondary mice (L4967 and L736). In total 151 mice were transplanted, with 122 showing engraftment in consecutive bone marrow punctures or in bone marrow harvests. CD10 sorts were performed on primary patient material (L784 and L49101) and on secondary samples harvested from engrafted primary mice (L4951, L8849, L2510 and L803) with 31 out of 52 mice transplanted with sorted material showing engraftment as seen with CD20 sorted cells. Blasts of all selected immunophenotypes were able to engraft the leukemia in unconditioned NSG mice as determined by 5 color flow cytometry. In particular, sorted cells of both fractions were able to reconstitute the complete phenotype of the leukemia. Harvested cells from engrafted mice could then be re-sorted into high and low antigen expressing fractions and successfully re-engrafted on secondary and tertiary mice. Cell purities of transplanted cells were usually higher than 90% (range 67-100%). The ability of all populations to serially engraft mice demonstrates long-term self-renewal capacity. Two additional patients were used in the limiting dilution assays (high WBC/t(4;11) high risk (L826); low WBC/MRD negative low risk (L792)) and experiments were performed on primary unsorted and secondary sorted material. Cell numbers necessary for ALL engraftment differed between individual leukemias but as little as 100 cells proved to be sufficient in one unsorted and in both the CD19+CD20low and CD19+CD20high fractions (Table 1). Mice transplanted with 10 cells only are still under observation. Table 1 Patient Transplant Population Cell dose Mice engrafted/transplanted L4951 Secondary CD20 high 500 3/3 CD20 low 3/3 CD20 high 100 3/3 CD20 low 3/3 L2510 Secondary CD20 high 3,000 2/4 CD20 low 4/4 CD20 high 300 0/4 CD20 low 1/4 L49101 Primary Unsorted 500 3/4 100 0/4 L792 Primary Unsorted 1,000 5/5 100 1/5 L826 Primary Unsorted 1,000 3/4 100 0/4 In conclusion we present strong evidence that leukemia-propagating cells are much more prevalent than previously thought and that blasts of diverse immunophenotype are able to serially reconstitute the complete leukemia in immune-deficient mice. Disclosures: No relevant conflicts of interest to declare.

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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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Acute Lymphocytic Leukemia and Down Syndrome

Blood ◽

10.1182/blood.v118.21.sci-8.sci-8 ◽

2011 ◽

Vol 118 (21) ◽

pp. SCI-8-SCI-8

Author(s):

Shai Izraeli

Keyword(s):

Down Syndrome ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Mtor Inhibitors ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Aberrant Expression ◽

Childhood All ◽

Increased Risk ◽

The Common

Abstract SCI-8 Children with Down syndrome are at a markedly increased risk for acute lymphoblastic leukemia (DS-ALL). These leukemias are exclusively of the B lymphoid precursor phenotype and occur in a similar age to “common” sporadic ALLs with the striking absence of infant ALL. Recent studies reveal that DS-ALLs are heterogeneous and differ from sporadic ALLs. Only about a fifth of DS-ALLs carry the common cytogenetic aberrations typical to childhood ALL. Genomic rearrangements leading to the expression of a cytokine receptor, CRLF2, are detected in 60% of DS-ALL in comparison with up to 10% of sporadic ALLs. CRLF2 heterodimerizes with Interleukin 7 receptor-α (IL7R) to form the receptor to thymic stromal lymphopoietin (TSLP). This receptor is usually present in macrophages, dendritic cells, and some T lymphocytes and participates in allergic and inflammatory processes. The aberrant expression of the TSLP receptor is DS-ALL (and sporadic ALL) is often associated with additional mutations that cause constitutive activation of the downstream JAK-STAT and mTOR growth signaling pathways. These are either lymphoid specific activating mutations of JAK2 or JAK1 or mutations in CRLF2 or IL7R that cause ligand-independent receptor dimerization. The role of the trisomy in selecting these somatic abnormalities is presently unknown. Clinically, the prognosis of DS-ALL is inferior to sporadic ALL mainly because of increased treatment toxicity. However, recent data suggest that the inferior outcome may also be related to the genetic properties of the leukemic cells and that excessive chemotherapy dose reduction may not be appropriate for these patients. Therefore increased vigilance for infectious complications and optimal supportive care are required during periods of intensive chemotherapy. The common activation of the TSLP signaling pathway in DS-ALLs suggests a future for targeted therapy with JAK and/or mTOR inhibitors. Importantly, research of DS-ALL has proven relevant for the general patient population with ALL, as somatic mutations in the TSLP pathway have been discovered in children and adults with sporadic ALL. A major research challenge is the elucidation of the roles of constitutional and somatic trisomy 21 in leukemogenesis. 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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