Leukemia Cell Xenograft in Ovo Enables Real-Time Testing of Response to Therapy

Abstract Background: Acute myeloid leukemia (AML) is most common in people over the age of 60 where it remains an almost incurable malignancy with a grim prognosis. Evaluation of new therapeutic agents in vitro and in vivo is critical for drug development, yet there are few in-vivo models for studying human leukemia and its therapy. The best model used is the high cost immune-deficient mice and that require several weeks to assess drug response. To complicate matters, AML almost certainly relapses with cells that are not necessarily exactly identical to the original malignant clone, often limiting therapeutic options. The development of anti-leukemia therapies could be facilitated by a rapid and cost effective in vivo system for evaluating response to new drugs. Additionally, decisions regarding personalized treatment for relapsed or refractory leukemia patients must be rapid, and produce results within several days, as longer time periods can be detrimental. Herein, we describe a fast, economical, in ovo turkey embryo model, which provides a unique system to meet these requirements. The model can be used for the assessment of human leukemia infiltration in medullary and extramedullary tissues and more importantly for rapid testing of anti- leukemic agents within the bone marrow (BM). This system can be applied for new drug development and for personalized real time response of patient cells to potential leukemia therapies. Methods: BCR/Abl+ AML lines K562 and LAMA-84 , c-Kit+ CHRF-4288, fresh AML patient and Raji Burkit lymphoma cells (5x106) were injected into turkey egg chorioallantoic membrane (CAM) veins on embryonic day E11 previously optimized (1). Engraftment in BM was detected by flow cytometry (FC) using anti-human CD71 or anti- human CD33 for AML and anti human CD45 for Raji cells, or by Quantitative real time PCR (Q-PCR) comparing the amount of genomic human to the amount of avian DNA and number of human /avian cells in BM. Drug response was tested by IV injection of therapeutic range doses of Imatinib (Glivec ®), Doxorubicin or dexamethasone, 48H after grafting cells. Drug levels were precalibrated to be non-toxic to the developing embryo by LD50 and BM cell viability compared to control (Taizi M et al Exp Hem 34:1698,2006, Grinberg I et al, Leuk Res. 33:1417, 2009). Six days later (E19) the embryos were sacrificed and the BM collected for FC and hematopoietic and non-hematopoietic tissues for molecular analysis. Results: The kinetics of leukemia cell engraftment in the BM on E15, E18, and E23 in BM and liver after cell injection on day E11 was assessed to determine the optimal treatment and readout times. The highest engraftment level in BM and liver was detected at E18 by Q-PCR, and FC in more than 90% of the injected embryos. We quantitatively compared the engraftment of AML cells at E18-20 without and with drug treatment that was administered IV 48 hours after cell injection. The average engraftment (±SD.) in the BM after one week was 4.5%+1.7 K562, 5.83% +0.88 LAMA-84, 11.2%+3.5 CHRF-4288, 8.9% +1.6 Raji (n=7-15 per group) and 2.5% fresh leukemia cells detected by FC and confirmed by Q-PCR. A single dose of either 0.75 mg Imatinib or 50 mg Doxorubicin /embryo previously calibrated to be non toxic to the embryos reduced engraftment of AML cells in BM and in several other organs by more than ten fold. A similar effect was also obtained by a single dose of 5mg dexamethasone in Raji injected embryos. Treatment with 0.5 mg Imatinib on injected ARH-77 (multiple myeloma) or Raji cells had no effect on cell engraftment, while treatment with a single non toxic dose of Revlimid as previously described eliminated engraftment of ARH77 cells (1), clearly demonstrating the specificity of the drugs and utility of the system. Conclusion: Our study demonstrates the potential utility of a practical and unique avian embryo model for testing drug activity on human AML cells in vivo. This system, under preclinical development, is expected to provide a new xenograft platform for real time affordable testing leukemia therapies. More importantly, this may open a new venue for individualized screening for response or resistance to specific therapeutic agents for the relapsed or refractive patient and may lead to better optimization of practical and applicable therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

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New Xenograft Method For Studying Leukemia and Drug Response In Turkey Embryos

Blood ◽

10.1182/blood.v122.21.3973.3973 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3973-3973

Author(s):

Varda R Deutsch ◽

Sigi Kay ◽

Yona Farnoushi ◽

Erez Matalon ◽

Tal Ohayon ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Single Dose ◽

Drug Response ◽

Cost Effective ◽

New Drugs ◽

Leukemia Cells ◽

Human Leukemia ◽

Cell Engraftment

Abstract Background A widely accepted in vivo model for studying leukemia and its treatment is the highly immune-deficient mice NOD/SCID (b2M-/- or rag-/-). While this model is powerful and recapitulates the phenotypes of blood malignancies in vivo. it is costly and complex, requiring 1-2 months to establish engraftment and the mice are susceptible to spontaneous neoplasms. For these and other reasons the testing of new drugs on leukemia is primarily performed in vitro. The development of antileukemia therapies could be facilitated by a rapid and cost-effective in vivo system for evaluating human leukemia growth and its response to new drugs. Additionally, the treatment of relapsed or refractory disease could be individually tailored by this rapid and cost-effective in vivo system by evaluating patient's cells response to new agents. Turkey embryos are inexpensive, require no maintenance, are larger than chicks are more easily manipulated and have a more robust engraftment (Grinberg I, et al, Leuk Res, 2009; 33:1417-26). We recently described this new in-vivo system for studying multiple myeloma in the pre-immune turkey embryo (Farnoushi, Y., et al.,Br J Cancer, 2011; 105:1708-18). We now demonstrate application of this rapid alternative xenograft system for the preclinical assessment of leukemia growth and therapy. Methods BCR/Abl+ human leukemia lines K562 or LAMA-84 c-Kit+ CHRF 4288 and fresh patient cells were injected into turkey egg chorioallantoic membrane (CAM) veins. Cell injections were performed on day embryonic day E11as previously optimized (Farnoushi, Y., et al.,as above). To determine the engraftment of human AML cells on E19-23, in hematopoietic tissue, the engraftment of human AML cells in the BM was detected in BM by flow cytometry (FC) using anti-human CD71 for LAMA and K562, anti- human CD33 for CHRF and fresh leukemia samples. Engraftment in bone marrow (BM) and other organs was also monitored using Quantitive real time PCR (Q-PCR) comparing the amount of genomic human to the amount of avian DNA and number of human cells / avian cells in BM. Drug response was tested by IV injection of therapeutic range doses of Imatinib (Glivec ®) and Doxorubicin, 48H after cell grafting, at drug levels precalibrated to be non-toxic to the developing embryo by LD50 and BM cell viability compared to control. Six days later (E19) the embryos were sacrificed and the BM collected for FC and hematopoietic and non-hematopoietic tissues for molecular analysis. Results The optimal treatment and readout times were resolved by injecting cells on E11 and determining the kinetics of leukemia cell engraftment in the BM on E15, E18, and E23 in BM and liver. The highest engraftment level in the BM bone marrow (BM) and liver of lines tested was detected at E18 by Q-PCR, and FC in more than 90% of the injected embryos. The average engraftment (±s.d.) in the BM after one week was 4.6%+0.75 K562, 5.16%+2.15 LAMA-84, 7.65%+1.15 CHRF-4288 ( n=7-12 per group) and 2.5% fresh leukemia cells was detected by FC. Q-PCR results were similar to those of FC. Imatinib toxicity testing revealed 100% survival of embryos with no BM toxicity on embryos treated on E13 with doses similar to a human therapeutic dose, up to 0.75 mg/egg. Treatment of embryos with 100 ug Doxorubicin was previously shown to be not toxic to the embryos (Taizi M et al. Exp Hematol 2006; 34:1698–708). A single dose of 0.75 mg Imatinib/embryo dramatically reduced engraftment in BM and several other organs of all 3 AML cell lines or fresh patient leukemia cells. A similar effect was also obtained by a single dose Rx 100ug Doxorubicin. Treatment of a single dose of 0.75 Imatinib mg/embryo 48H after injecting ARH-77 (multiple myeloma) had no effect on cell engraftment. Treatment with a single non toxic dose of Revlimid as previously described (Farnoushi, Y., et al. as above) eliminated engraftment of ARH77 cells, clearly demonstrating the specificity of the drug treatments. Conclusions The results presented demonstrate the potential utility of a practical avian embryo model for testing drug activity in vivo. With further work the turkey embryo may provide a new xenograft in vivo method for studying the biology of leukemia engraftment, and for rapidly and affordably testing leukemia therapies. This system may provide a new platform for developing individualized patient screening for response or resistance to particular therapeutic agents. Disclosures: No relevant conflicts of interest to declare.

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Curcumin and Its Analogue Induce Apoptosis in Leukemia Cells and Have Additive Effects with Bortezomib in Cellular and Xenograft Models

BioMed Research International ◽

10.1155/2015/968981 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 16

Author(s):

L. I. Nagy ◽

L. Z. Fehér ◽

G. J. Szebeni ◽

M. Gyuris ◽

P. Sipos ◽

...

Keyword(s):

Leukemia Cell ◽

Leukemic Cells ◽

Leukemia Cell Line ◽

Leukemia Cells ◽

Great Promise ◽

Human Leukemia ◽

Human Leukemia Cell Line ◽

Apoptotic Effects

Combination therapy of bortezomib with other chemotherapeutics is an emerging treatment strategy. Since both curcumin and bortezomib inhibit NF-κB, we tested the effects of their combination on leukemia cells. To improve potency, a novel Mannich-type curcumin derivative, C-150, was synthesized. Curcumin and its analogue showed potent antiproliferative and apoptotic effects on the human leukemia cell line, HL60, with different potency but similar additive properties with bortezomib. Additive antiproliferative effects were correlated well with LPS-induced NF-κB inhibition results. Gene expression data on cell cycle and apoptosis related genes, obtained by high-throughput QPCR, showed that curcumin and its analogue act through similar signaling pathways. In correlation with in vitro results similar additive effect could be obsereved in SCID mice inoculated systemically with HL60 cells. C-150 in a liposomal formulation given intravenously in combination with bortezomib was more efficient than either of the drugs alone. As our novel curcumin analogue exerted anticancer effects in leukemic cells at submicromolar concentration in vitro and at 3 mg/kg dose in vivo, which was potentiated by bortezomib, it holds a great promise as a future therapeutic agent in the treatment of leukemia alone or in combination.

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Targeting the Hedgehog Signaling Pathway in Therapy-Resistant BCR-ABL1 Positive Leukemia with Ponatinib

Blood ◽

10.1182/blood.v118.21.63.63 ◽

2011 ◽

Vol 118 (21) ◽

pp. 63-63

Author(s):

Seiichiro Katagiri ◽

Tetsuzo Tauchi ◽

Seiichi Okabe ◽

Eishi Ashihara ◽

Shinya Kimura ◽

...

Keyword(s):

Hedgehog Signaling ◽

Kinase Inhibitor ◽

Leukemia Cell ◽

Hedgehog Pathway ◽

Leukemia Cell Line ◽

Human Leukemia ◽

Combined Effects ◽

Human Leukemia Cell Line ◽

Induction Of Apoptosis

Abstract Abstract 63 The hedgehog signaling pathway is a key regulator of cell growth and differentiation during development. While the hedgehog pathway is inactive in most normal adult tissues, hedgehog pathway reactivation has been implicated in the pathogenesis of several neoplasms. Recent studies demonstrated that hedgehog pathway is involved in the development of B cell acute lymphoblastic leukemia (B-ALL), as well as self-renewal and survival of B-ALL. Vismodegib is a selective hedgehog pathway inhibitor that blocks hedgehog signaling by binding to Smo and inhibiting activation of downstream hedgehog target genes. In the present study, we investigated the combined effects of vismodegib and ponatinib, a pan-ABL1 kinase inhibitor, in mutant forms of BCR-ABL1-expressing BaF3 cells and T315I-expressing human leukemia cell line, SK-9 (Exp Hematol. 2010; 38:765). We observed that the treatments with sonic hedgehog (Shh) enhanced the proliferation of SK-9 cells, correlated with the up regulation of Cyclin D2 and Bcl-2. The treatment with Shh significantly reduced the induction of apoptosis in ponatinib-treated SK-9 cells, however, co-tratment with vismodegib and ponatinib resulted in significantly more induction of apoptosis in Shh-treated SK-9 cells. Combined treatment with vismodegib and ponatinib in SK-9 cells also associated with the reduction of Cyclin D2 and Bcl-2, and more PARP cleavage, resulting from increased activation of caspase-3 and -9 during apoptosis. We next conducted the experiments to further evaluate the mechanism of cooperation between vismodegib and ponatinib in SK-9 cells. SK-9 cells were transfected with control siRNA or Smo siRNA or Gli1 siRNA. At 48 h after transfection, Shh co-cultured SK-9 cells were treated with indicated concentration of ponatinib for 48 h, and viable cells were counted. In the presence of Smo siRNA or Gli1 siRNA, SK-9 cells increased antiproliferative activity with ponatinib. These results demonstrated that hedgehog signaling activation impairs the efficacy of pan-ABL1 kinase inhibitor. To assess the in vivo efficacy of ponatinib and vismodegib, athymic nude mice were injected s.c. with BaF3 cells expressing wild-type (WT)-BCR-ABL1 and BCR-ABL1 mutants (M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). 5 days after injection (average tumor volume, 100 mm3), the mice were randomized into four groups (5 mice per group), with each group receiving either vehicle, ponatinib (30 mg/kg; q.d.), vismodegib (10 mg/kg; q.d.), ponatinib (30 mg/kg; q.d.) + vismodegib (10 mg/kg; q.d.). The ponatinib and vismodegib combination more effectively inhibited tumor growth in mice compared to either vehicle- or ponatinib- or vismodegib-treated mice. Histopathologic analysis of tumor tissue from ponatinib + vismodegib-treated mice demonstrated an increased number of apoptotic cells detected by TUNEL stain. To investigate combined effects of vismodegib and ponatinib on T315I-expressing human leukemia cell line, NOD/SCID mice were injected intravenously with SK-9 cells. Treatment with vismodegib and ponatinib demonstrated a marked segregation of apoptotic cells in both the central bone-marrow cavity, the endosteal surface, spleen and liver. These results suggest that the combination with a Smo inhibitor and ABL1 tyrosine kinase inhibitors (TKIs) may help to eliminate the therapy-resistant T315I BCR-ABL1 positive ALL cells. In summary, our preclinical results indicate that vismodegib has potential as an important option for controlling minimal residual cells in BCR-ABL1-positive ALL. The combined results of cell-based, and in vivo studies suggest that vismodegib exhibits sufficient activity against mutants form of BCR-ABL1 to warrant consideration for combined use with pan-ABL1 TKIs. Disclosures: No relevant conflicts of interest to declare.

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Targeting of Chronic Lymphocytic Leukemia B Cells with a Novel Monoclonal Antibody to ROR1

Blood ◽

10.1182/blood.v118.21.984.984 ◽

2011 ◽

Vol 118 (21) ◽

pp. 984-984

Author(s):

Bing CUi ◽

George F. Widhopf ◽

Jian Yu ◽

Daniel Martinez ◽

Esther Avery ◽

...

Keyword(s):

Monoclonal Antibody ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Adoptive Transfer ◽

Leukemia Cell ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Human Leukemia ◽

Phosphorylated Akt

Abstract Abstract 984 ROR1 is an orphan receptor tyrosine kinase that is expressed on leukemia cells of patients with chronic lymphocytic leukemia (CLL), but not on most adult tissues of healthy adults, including CD5+ B cells. To generate anti-ROR1 antibodies, we immunized mice using different strategies employing vaccines comprised of recombinant ROR1 protein, polynucleotide-ROR1 vaccines and CD154 genetic adjuvants, or replication-defective adenovirus vectors encoding ROR1 and CD154. We extirpated the spleens of animals that developed high-titer serum anti-ROR1 antibodies and used these to generate monoclonal-antibody-(mAb)-producing hybridomas or antibody phage-display libraries that subsequently were screened for ROR1-binding. Over 70 unique mAbs were generated that each bound the extra-cellular domain of native ROR1. Most mAbs recognized an epitope(s) within the ROR1 Ig-like domain, which appears to represent the immune dominant epitope. Other mAb recognized epitopes within the conserved ROR1 Kringle domain. One mAb (UC D10-001) had distinctive binding to an intradomain epitope of human ROR1 (hROR1). UC D10-001 was the only mAb we found directly cytotoxic for hROR1-expressing leukemia cells cultured in media without complement for 6 hours. We found that UC D10-001 could induce significant reductions in basal levels of phosphorylated AKT in hROR1-expressing leukemia cells. Moreover, UC D10-001 significantly decreased the basal levels of phosphorylated AKT in freshly isolated human CLL cells (N=4) to levels comparable to that observed in co-cultures containing 10 mM LY294002, a broad-spectrum inhibitor of PI3K. We examined whether this mAb had cytotoxic activity for leukemia cell in vivo. For this we examined whether we could inhibit the adoptive transfer of human-ROR1-expressing leukemia cells to young, syngeneic recipient mice made transgenic for human ROR1 under control of a B-cell specific promoter. Cohorts of 5 animals per group were each given intravenous injections of antibody at a dose of at 10 mg/kg. Each cohort was treated with UC D10-001, control IgG, or 4A5, an anti-ROR1 mAb specific for a non-cross-reactive epitope located in the Ig-like domain of ROR1. Each animal received an intravenous injection of 5 × 105 ROR1-expressing leukemia cells and then was assessed weekly for circulating leukemia cells by flow cytometry. UC D10-001, but not control IgG or 4A5, significantly inhibited engraftment of the ROR1+ leukemia. Four weeks after adoptive transfer, animals treated with UC D10-001 had a 10-fold lower median number of leukemia B cells in the blood than animals treated with control IgG or 4A5. We also tested UC D10-001 for its capacity to induce clearance of human ROR1+ CLL cells engrafted into the peritoneal cavity of Rag-2−/−/γc−/− immune deficient mice. Each of these mice received intraperitoneal injections of equal numbers of human ROR1+ CLL cells prior to receiving D10-001, control IgG, or 4A5, each at 10 mg/kg. These animals were sacrificed seven days later and the human leukemia cells were harvested via peritoneal lavage. In mice treated with UC D10-001 we harvested an average of only 6 × 104 ± 3 × 104 CLL cells. This number of cells was significantly less than the average number of CLL cells harvested from control IgG or 4A5-treated mice (8 × 105 ± 4 × 105 or 7 × 105 ± 2 × 105, respectively, p <0.01). These studies indicate that the anti-ROR1 mAb UC D10-001 can be directly cytotoxic for ROR1-expressing leukemia cells in vitro and in vivo, a property that apparently is unique to this mAb among other anti-ROR1 mAbs. Because of the restricted expression of ROR1 on leukemia cells and the distinctive properties of this mAb, we propose that UC D10-001 might have potential utility in the treatment of patients with CLL. Disclosures: No relevant conflicts of interest to declare.

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Wnt5a Induces Association of ROR1 with 14-3-3ζ to Enhance Chemotaxis and Proliferation in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v128.22.349.349 ◽

2016 ◽

Vol 128 (22) ◽

pp. 349-349 ◽

Cited By ~ 1

Author(s):

Jian Yu ◽

Liguang Chen ◽

Yun Chen ◽

Ling Zhang ◽

Laura Z. Rassenti ◽

...

Keyword(s):

Mass Spectrometry ◽

Research Funding ◽

Critical Role ◽

Leukemia Cell ◽

Lymphocytic Leukemia ◽

Orphan Receptor ◽

Binding Motif ◽

Human Leukemia

Abstract Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic antigen that is expressed on CLL cells, but not on normal postpartum tissues. We found that ROR1 was a receptor for Wnt5a, which could activate Rho GTPases (e.g. RhoA and Rac1) in CLL cells by inducing the recruitment to ROR1 of guanine exchange factors (GEFs), notably ARHGEF2. How ARHGEF2 can complex with ROR1 was not known. We performed mass spectrometry-based proteomics to interrogate immune-precipitates of Wnt5a-activated ROR1 and identified 14-3-3ζ, a highly conserved, cytoplasmic-protein member of the tetratricopeptide repeat-like superfamily. 14-3-3ζ plays a critical role in cell-signaling pathways, which promote proliferation, adhesion, and survival in a variety of human cancers. We validated the Wnt5a-induced interaction of ROR1 with 14-3-3ζ in primary CLL cells using co-immunoprecipitation studies and immunoblot analyses. We found the capacity of Wnt5a to induce ROR1 to associate with 14-3-3ζ could be blocked by cirmtuzumab, a first-in-class humanized mAb specific for a functional epitope in the ROR1-extracellular domain; this mAb is undergoing clinical testing in patients with CLL. Furthermore, we found that 14-3-3ζ could interact with ARHGEF2 in CLL cells. Silencing 14-3-3ζ via RNAi impaired the capacity of Wnt5a to: (1) induce recruitment of ARHGEF2 to ROR1, (2) enhance the in vitro exchange activity of ARHGEF2 for RhoA and Rac1, and (3) induce activation of RhoA and Rac1 in primary CLL cells. Consistent with these findings, we found that Difopein, an inhibitor of 14-3-3ζ, also could inhibit Wnt5a-enhanced chemokine-directed migration and proliferation of primary CLL cells in vitro, at low concentrations that did not result in leukemia-cell apoptosis. To examine structure-function relationships, we employed the MEC1 cell line, which was derived from human CLL. Prior studies found MEC1 cells expressed Wnt5a, which induced activation of RhoA and Rac1 in MEC1 cells made to express ROR1 (MEC1-ROR1), but not in parental MEC1 cells, which lacked ROR1. Similar to work on primary CLL cells, we detected 14-3-3ζ in anti-ROR1 immune precipitates via mass spectrometry and immunoblot analyses. We identified a 14-3-3ζ binding motif (RSPS857SAS) in the cytoplasmic domain of ROR1; site directed mutagenesis and transfection of MEC1 with mutant forms of ROR1 determined that serine-857 was required for the recruitment of 14-3-3ζ and ARHGEF2-dependent activation of RhoA and Rac1, respectively. In addition, we used CRISPR/Cas9 technology to delete 14-3-3ζ (Δ14-3-3ζ) in MEC1 and MEC1-ROR1 cells. We found that MEC1-ROR1 had significantly higher rates of proliferation than MEC1-ROR1-Δ14-3-3ζ cells, which in turn had rates of proliferation comparable to those of MEC1 cells or MEC1-Δ14-3-3ζ lacking expression of ROR1. MEC1-ROR1 also had a significantly greater capacity to migrate in response to chemokine (CCL21) than did MEC1-ROR1-Δ14-3-3ζ cells, which migrated in response to CCL21 as well as MEC1-Δ14-3-3ζ or MEC1 cells lacking expression of ROR1. To examine whether such differences affected leukemia-cell growth in vivo, we engrafted Rag2−/−γc−/− mice each with equal numbers of MEC1, MEC1-Δ14-3-3ζ, MEC1-ROR1, or MEC1-ROR1-Δ14-3-3ζ cells. We found that mice that received MEC1-ROR1 cells had significantly higher levels of engraftment and human leukemia-cell proliferation than did mice that received MEC1, MEC1-Δ14-3-3ζ, or ROR1-Δ14-3-3ζ cells, which had comparable levels of engraftment and proliferation, indicating that 14-3-3ζ was necessary for the engraftment/growth advantage of MEC1-ROR1 over MEC1 cells in vivo. Collectively, this study reveals that 14-3-3ζ plays a critical role in Wnt5a/ROR1-dependent-signaling leading to enhanced migration and proliferation of CLL cells in vitro and in vivo. Disclosures Kipps: Roche: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding.

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Proliferation and differentiation of human myeloid leukemic cells in immunodeficient mice: electron microscopy and cytochemistry

Blood ◽

10.1182/blood.v63.5.1015.1015 ◽

1984 ◽

Vol 63 (5) ◽

pp. 1015-1022 ◽

Cited By ~ 17

Author(s):

EA Machado ◽

DA Gerard ◽

CB Lozzio ◽

BB Lozzio ◽

JR Mitchell ◽

...

Keyword(s):

Nude Mice ◽

Leukemia Cell ◽

Leukemic Cells ◽

Leukemia Cell Line ◽

Leukemia Cells ◽

Human Leukemia ◽

Immunodeficient Mice ◽

Human Leukemia Cells

Abstract To study the influence of a biologic environment on cultured human leukemia cells, KG-1, KG-1a, and HL-60 cells were inoculated subcutaneously into newborn nude mice. The cells developed myelosarcomas at the site of inoculation and in lungs and kidneys. KG-1 and HL-60 myelosarcomas were successfully passaged through adult nude mice, whereas KG-1a tumors proliferated only after transplantation into newborn hosts. The human nature of the cells forming myelosarcomas in mice was assessed by chromosomal analyses and detection of cross- reactivity with an antibody to the human leukemia cell line K562. We undertook electron microscopic and cytochemical examinations of the cells proliferating in vitro and in the mice. The granules of KG-1 cells in vivo did not react for acid phosphatase, as observed in vitro, and the HL-60 cells proliferating in mice lost the perinuclear myeloperoxidase (MPO) demonstrated in cultured cells. Although the influence of an in vivo selection of cell subpopulations cannot be ruled out, the enzymatic changes are compatible with induced cell differentiation. Conclusive evidence of differentiation in vivo was observed in the KG-1a cell subline. The undifferentiated KG-1a blasts developed cytoplasmic granules and synthesized MPO during proliferation in vivo. These observations indicate that human leukemia cells from established cell lines proliferate in nude mice and may acquire new differentiated properties in response to the in vivo environment.

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A Novel N-Oxide Drug, AQ4N, Has In Vitro Activity in Lymphoma and Leukemia Cell Lines and Selectively Targets Lymphocytes and Lymphatic Tissues in Vivo.

Blood ◽

10.1182/blood.v104.11.2498.2498 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2498-2498

Author(s):

Jeffrey L. Cleland ◽

Alvin Wong ◽

Susan E. Alters ◽

Peter A. Harris ◽

Chris R. Dunk ◽

...

Keyword(s):

Cell Lines ◽

Leukemia Cell ◽

Lymphoid Tissues ◽

Human Leukemia ◽

Repeat Dose ◽

Cynomolgus Monkeys ◽

Leukemia Cell Lines ◽

Human Lymphoma

Abstract An ideal treatment for lymphoma and leukemia is the use of highly selective compounds to eliminate diseased cells with minimal systemic toxicity to normal tissues (cf. imatinib mesylate; Gleevec). AQ4N (1,4 bis[[2-(dimethylamino)ethylamino}-5,8-hydroxyanthracene-9,10-dione bis N-oxide) is designed to have little or no toxicity until selectively activated by bioreduction in hypoxic cells to AQ4 (reduced AQ4N), a highly potent DNA topoisomerase II inhibitor. In a series of studies, AQ4 has been shown to have potent cytotoxicity on lymphoma and leukemia cell lines in vitro and AQ4N has selective activity in lymphatic tissues in vivo. The IC50 of AQ4, was 0.63, 12.0, 90.5 and 150 nM in Namalwa, Daudi, Ramos, and Raji human lymphoma cell lines and 1.0, 6.0, and 20 nM in HL-60, KG1a and K562 human leukemia cell lines. On several of the tumor lines the activity of AQ4 was more potent than doxorubicin (i.e. IC50 for Dox was 20.3 nM on Namalwa). AQ4N also had anti-proliferative activity at μM levels indicating a potential mechanism for activation by these cell lines. In repeat dose toxicology studies of AQ4N in pigmented rats and cynomolgus monkeys, the maximum tolerated doses (MTD; rats: 20 mg/kg/wk x 6; monkeys 6 mg/kg/wk x 6) resulted in lymphoid tissue atrophy. A decrease in lymphocyte levels and atrophy of the spleen, thymus, and mandibular and mesenteric lymph nodes were observed at terminal sacrifice of the animals. In contrast, there was an absence of myelosuppression and only mild neutropenia and minor bone marrow atrophy at the MTD. Administration of radiolabeled AQ4N (14C-benzene) to pigmented rats and cynomolgus monkeys indicated persistence of AQ4N radioactivity in lymphoid tissues for several weeks after a single dose (rats: 20 mg/kg (130–140 μCi/kg); monkeys: 10 mg/kg (135 μCi/kg)). For example, in rats the half-life of radioactive AQ4N in the spleen was 538 hrs with 0.9 μg AQ4N/g tissue (spleen) remaining one week after dosing. Monkeys demonstrated a similar effect with 76.5–86.8 μg AQ4N/g tissue observed in the spleen one week after treatment. Other tissues contained significantly less radioactive AQ4N with the exception of the liver (67.9–78.6 μg AQ4N/g tissue) and adrenal cortex (78.7–86.6 μg AQ4N/g tissue). While some hypertrophy and eosinophila was observed in the adrenal glands, liver toxicity was not observed at the MTD in the repeat dose cynomolgus monkey toxicology study. Overall, these initial findings indicate that AQ4N is active in vitro against human lymphoma and leukemia cell lines and selectively targets lymphoid tissues in vivo suggesting the potential benefit of AQ4N in the treatment of lymphoproliferative diseases.

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Adipocytes Secrete Factors That Cause Leukemia Drug Resistance.

Blood ◽

10.1182/blood.v112.11.1346.1346 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1346-1346

Author(s):

James W Behan ◽

Jason P Yun ◽

Marina P Proektor ◽

Ehsan A Ehsanipour ◽

Anna Butturini ◽

...

Keyword(s):

Bone Marrow ◽

Chemotherapy Resistance ◽

Leukemia Cell ◽

Conditioned Media ◽

Leukemia Cells ◽

Cell Contact ◽

Human Leukemia ◽

Bone Marrow Niche

Abstract We have previously shown that obesity is an independent predictor of leukemia relapse in children. We have also shown that obese mice transplanted with syngeneic leukemia cells have poorer survival after chemotherapy, even when they are dosed proportional to body weight. Since interactions between leukemia cells and cells of the bone marrow niche are considered important for chemotherapy resistance and relapse, and adipocytes can comprise ~50% of the bone marrow niche, we developed in vivo and in vitro models to investigate the role of adipocytes in the leukemia microenvironment. Obese C57Bl/6J mice were transplanted with GFP+ murine preB cell ALL (“8093”) cells and then treated with vincristine (0.5 mg/kg/week × 3 weeks). At the time of relapse, we found that GFP+ leukemia cells persisted in the fat pads of the mice. We then developed an in vitro co-culture system in which human or murine leukemia cells were cultured together with adipocytes (differentiated 3T3-L1s). Undifferentiated 3T3-L1 cells, which are fibroblastic in nature, were used as a control. In this model, adipocytes severely diminished the anti-leukemic effect of all chemotherapeutics tested against murine 8093 cells, including vincristine, dexamethasone, nilotinib, daunorubicin, and L-asparaginase. Adipocytes also protected murine T-cell ALL and human SD-1, RCH-ACV, and BV173 cells from vincristine and daunorubicin. Adipocyte protection of leukemia cells occurred independent of cell contact. Further experiments demonstrated that media conditioned by adipocytes was able to protect 8093 cells from a 3-day exposure to 25 nM dexamethasone (viable cells were at 40±12% of their plated value in regular media, 66±17% in fibroblast-conditioned media, and 109±24% in adipocyte-conditioned media, p<0.05). Surprisingly, adipocyte-conditioned media did not protect leukemia cells from daunorubicin. However, media conditioned by the presence of both adipocytes and leukemia cells simultaneously conferred a high degree of resistance to the leukemia cells (n=3, p<0.05 compared to all other media types). In summary, adipose tissue is a reservoir for relapsed leukemia cells in vivo. Adipocytes engender protection from multiple chemotherapies in murine and human leukemia cell lines. Adipocytes secrete factor(s) that confer dexamethasone and daunorubicin resistance to leukemia cells, though for the latter drug it appears that a two-way communication between leukemia and adipocytes may be necessary for this protection. Figure Figure

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A small molecule screening strategy with validation on human leukemia stem cells uncovers the therapeutic efficacy of kinetin riboside

Blood ◽

10.1182/blood-2011-01-330019 ◽

2012 ◽

Vol 119 (5) ◽

pp. 1200-1207 ◽

Cited By ~ 23

Author(s):

Sean P. McDermott ◽

Kolja Eppert ◽

Faiyaz Notta ◽

Methvin Isaac ◽

Alessandro Datti ◽

...

Keyword(s):

Stem Cells ◽

Regulatory Networks ◽

Leukemia Cell ◽

Screening Strategy ◽

Comparable Efficacy ◽

Human Leukemia ◽

Hematopoietic Stem ◽

Novel Approach

Abstract Gene regulatory networks that govern hematopoietic stem cells (HSCs) and leukemia-initiating cells (L-ICs) are deeply entangled. Thus, the discovery of compounds that target L-ICs while sparing HSC is an attractive but difficult endeavor. Presently, most screening approaches fail to counter-screen compounds against normal hematopoietic stem/progenitor cells (HSPCs). Here, we present a multistep in vitro and in vivo approach to identify compounds that can target L-ICs in acute myeloid leukemia (AML). A high-throughput screen of 4000 compounds on novel leukemia cell lines derived from human experimental leukemogenesis models yielded 80 hits, of which 10 were less toxic to HSPC. We characterized a single compound, kinetin riboside (KR), on AML L-ICs and HSPCs. KR demonstrated comparable efficacy to standard therapies against blast cells in 63 primary leukemias. In vitro, KR targeted the L-IC–enriched CD34+CD38− AML fraction, while sparing HSPC-enriched fractions, although these effects were mitigated on HSC assayed in vivo. KR eliminated L-ICs in 2 of 4 primary AML samples when assayed in vivo and highlights the importance of in vivo L-IC and HSC assays to measure function. Overall, we provide a novel approach to screen large drug libraries for the discovery of anti–L-IC compounds for human leukemias.

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Clinical MDR1 inhibitors enhance Smac-mimetic bioavailability to kill murine LSCs and improve survival in AML models

Blood Advances ◽

10.1182/bloodadvances.2020001576 ◽

2020 ◽

Vol 4 (20) ◽

pp. 5062-5077

Author(s):

Emma Morrish ◽

Anthony Copeland ◽

Donia M. Moujalled ◽

Jason A. Powell ◽

Natasha Silke ◽

...

Keyword(s):

Leukemia Cell ◽

Leukemic Cells ◽

Human Leukemia ◽

Combination Strategy ◽

Multidrug Resistance Protein 1 ◽

Leukemia Cell Lines ◽

Therapeutic Opportunity ◽

Smac Mimetic ◽

Approved Drugs

Abstract The specific targeting of inhibitor of apoptosis (IAP) proteins by Smac-mimetic (SM) drugs, such as birinapant, has been tested in clinical trials of acute myeloid leukemia (AML) and certain solid cancers. Despite their promising safety profile, SMs have had variable and limited success. Using a library of more than 5700 bioactive compounds, we screened for approaches that could sensitize AML cells to birinapant and identified multidrug resistance protein 1 inhibitors (MDR1i) as a class of clinically approved drugs that can enhance the efficacy of SM therapy. Genetic or pharmacological inhibition of MDR1 increased intracellular levels of birinapant and sensitized AML cells from leukemia murine models, human leukemia cell lines, and primary AML samples to killing by birinapant. The combination of clinical MDR1 and IAP inhibitors was well tolerated in vivo and more effective against leukemic cells, compared with normal hematopoietic progenitors. Importantly, birinapant combined with third-generation MDR1i effectively killed murine leukemic stem cells (LSCs) and prolonged survival of AML-burdened mice, suggesting a therapeutic opportunity for AML. This study identified a drug combination strategy that, by efficiently killing LSCs, may have the potential to improve outcomes in patients with AML.

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