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. Author manuscript; available in PMC: 2014 Aug 7.
Published in final edited form as: Leukemia. 2011 Dec 20;26(5):985–990. doi: 10.1038/leu.2011.360

Inhibition of CXCR4 in CML cells disrupts their interaction with the bone marrow microenvironment and sensitizes them to nilotinib

Potentiation of nilotinib by CXCR4 antagonist

Ellen Weisberg 1,*, Abdel Kareem Azab 1,*, Paul W Manley 2, Andrew L Kung 3, Amanda L Christie 3, Rod Bronson 4, Irene M Ghobrial 1,5, James D Griffin 1,5
PMCID: PMC4124489  NIHMSID: NIHMS582723  PMID: 22182920

Abstract

Drug resistance is a growing area of concern. It has been shown that a small, residual pool of leukemic CD34+ progenitor cells can survive in the marrow microenvironment of chronic myeloid leukemia (CML) patients after years of kinase inhibitor treatment. Bone marrow stroma has been implicated in the long-term survival of leukemic cells, and contributes to the expansion and proliferation of both transformed and normal hematopoietic cells. Mechanistically, we found that CML cells expressed CXCR4, and that plerixafor diminished BCR-ABL-positive cell migration and reduced adhesion of these cells to extra-cellular-matrix components and to bone marrow stromal cells in vitro. Moreover, plerixafor decreased the drug resistance of CML cells induced by co-culture with bone marrow stromal cells in vitro. Using a functional mouse model of progressive and residual disease, we demonstrated the ability of the CXCR4 inhibitor, plerixafor, to mobilize leukemic cells in vivo, such that a plerixafor-nilotinib combination reduced the leukemia burden in mice significantly below the baseline level suppression exhibited by a moderate-to-high dose of nilotinib as single agent. These results support the idea of using CXCR4 inhibition in conjunction with targeted tyrosine kinase inhibition to override drug resistance in CML and suppress or eradicate residual disease.

Introduction

In clinical trials, among CML patients who discontinue imatinib therapy after having maintained a complete molecular response (≥5 log reduction in BCR-ABL transcripts) for two years, the disease reemerges in 61% of cases1. Since BCR-ABL-expressing leukemic stem cells persist in the bone marrow (BM) of CML patients with sustained undetectable molecular residual disease treated with IFN-alpha, imatinib, or dasatinib,2 such cell populations might be responsible for the reemergence of disease following the cessation of therapy. Although isolated leukemic CD34+ cells have been shown to be insensitive to BCR-ABL tyrosine kinase inhibitors3, BM stromal cells, which provide survival signals that protect leukemic cells from inhibitor effects, have also been implicated in progenitor cell resistance2. Indeed, the quantity of leukemic stem cells that rely on stroma to survive has been found to be predictive of disease outcome4. Thus a better understanding of the role of the BM microenvironment in CML and its association with drug resistance is needed.

BM stroma and stroma-derived factors are thought to play a role in the long-term survival and growth of normal and leukemic cells in CML and other hematological malignancies, such as multiple myeloma and lymphoma5-16. BM stroma derived granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF) and stromal cell-derived factor-1α (SDF1α), either prevent terminal differentiation of hematopoietic stem cells or support their proliferation17-23. Splenic stroma has also been implicated in increased survival of both normal and leukemic cells24-25.

We previously demonstrated the stromal protection of leukemic cells from the anti-proliferative effects of nilotinib, and identified stromal-derived viability factors, including IL-6 and GM-CSF, as possibly mediating protection of tyrosine kinase inhibitor-treated leukemic cells26. Additionally, in nilotinib-treated mice we found high leukemia burden in tissues having ample sources of hematopoiesis-promoting stroma, suggesting that such tissues might support normal and malignant hematopoietic stem cell development. These studies revealed a leukemia distribution pattern consistent with that observed in imatinib- or nilotinib-treated patients.

A potential strategy to override stromal-mediated chemoresistance is to employ antagonists of the chemokine stromal cell-derived factor 1 (SDF-1) receptor, CXCR4, which mediates the migration of hematopoietic cells to the BM and plays a key role in leukemic cell-stromal cell interactions. Of relevance, in BCR-ABL positive cells CXCR4 expression is down-regulated and CXCR4 signaling is impaired27,29. BCR-ABL inhibition with either imatinib or nilotinib, can up-regulate the surface expression of CXCR4 to induce leukemic cell migration to the BM microenvironment leading to stroma-mediated chemoresistance of quiescent CML progenitor cells27-29. CXCR4 antagonists may be effective in enhancing kinase inhibitor-induced apoptosis of stromal-protected leukemic cells, implicating a causal relationship between the chemokine receptor CXCR4 and SDF-1α interaction and drug-resistant leukemia30. Indeed, studies have shown that the CXCR4 antagonist plerixafor (AMD3100; Genzyme)31, is effective in enhancing chemotherapy- or tyrosine kinase inhibitor-induced apoptosis of BM stroma-protected acute myeloid leukemia (AML)32 and multiple myeloma12,33.

The potentiating effects of plerixafor in reducing stroma-associated minimal residual disease following tyrosine kinase inhibition in CML have been explored to a lesser extent than other hematological malignancies in vivo. The studies presented here are demonstrate the ability of plerixafor to both lower leukemia burden and prolong survival of ABL nilotinib-treated mice harboring BCR-ABL-positive disease.

Materials and Methods

Cell lines and cell culture

BCR-ABL tyrosine kinase dependent murine 32D.p210 cells were developed as described34. The human CML cell-lines, K562 and KU812, were purchased from American Type Culture Collection (Rockville, MD). All cell-lines were cultured with 5% CO2 at 37°C in RPMI (Mediatech, Inc., Herndon, VA) with 10% fetal calf serum (FCS) and supplemented with 1% L-glutamine. Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and cultured according to manufacturer's instructions (Walkersville, MD).

CML patient bone marrow stroma

Human bone marrow cells were obtained from a newly diagnosed chronic phase CML patient after obtaining informed consent on an institutional IRB approved protocol. Mononuclear cells were isolated by density gradient centrifugation through Ficoll-Plaque Plus (Amersham Pharmacia Biotech AB, Uppsala, Sweden) at 2000 rpm for 30 min, followed by two washes in 1× PBS. Cells were plated in DMEM media containing 20% FBS and stroma was monitored for growth over a span of several weeks.

Flow cytometry

CML cell lines (KU812F and KS62,) were washed with cold PBS, blocked for 30 min with 5% FBS in PBS on ice, and treated with APC-conjugate of mouse anti-human –CXCR4 monoclonal antibody (5μg/ml, clone 12G5), or with an isotype control, for 1 hr on ice. Cells were then washed and analyzed by BD-Canto flow-cytometer.

Adhesion assays

An in vitro adhesion assay coated with fibronectin, endothelial cells and BMSCs was performed as previously described12. Briefly, BMSCs or HUVECs were cultured overnight to confluence in 96-well plates (5×103 cells/well) before initiating the adhesion assay. KU812F cells were treated with plerixafor (50 μM for 3 hr) before performing the adhesion assay, pre-labeled with Calcein-AM (Invitrogen Carlsbad, CA), added to plates coated with fibronectin (EMD, Gibbstown, NJ) with HUVECs or with BMSCs (1×105 cells/well), and allowed to adhere for 2 hr at 37°C. Non-adherent cells were washed away, and adherent cells were detected by measuring fluorescence intensity in the wells using a fluorescent-plate reader (Ex/Em=485/520 nm).

Trans-well and Trans-endothelial migration assay

Migration was determined by using the transwell migration assay (Corning Life Sciences, Acton, MA), according to the manufacturer's instructions and as previously described12. Briefly, KU812F cells were treated with 0 or 50 μM of plerixafor for 3 hr prior to carrying out the migration assay. KU812F cells were added to the upper chamber of the basket (2×105 cells/well), and left to migrate (for 4 hr at 37°C) towards the lower chamber, which contained no SDF1α or 30 nM of SDF1α. In some cases, HUVECs (5×103 cells/basket) were incubated overnight in the upper chamber of an eight-micron pore filters (Costar, NY) before performing the adhesion assay for trans-endothelial migration assay. Those cells that migrated to the lower chambers were counted by flow cytometry.

The effect of stroma on drug resistance

BMSCs (2×104 cells/well) were plated overnight in 24-well plates. KU812F cells (2×105 cells/ml) were treated with plerixafor (50 μM), and after 3 hr were cultured with or without BMSCs with the presence or absence of plerixafor. After 3 hr, cells were treated with 0 or 5 nM or nilotinib, and kept in culture for 24 hr. KU812F cells were then separated from the BMSCs, washed, and stained with FITC-Annexin-V and analyzed by flow cytometry.

Bioluminescent BCR-ABL model of CML

32D.p210 cells were transduced with a retrovirus encoding firefly luciferase (MSCV-Luc), and selected with G418 at a concentration of 1 mg/mL to produce the 32D.p210-luciferase (luc+) cell-line. Bioluminescence imaging was carried out as previously described35. Briefly, virus- and Mycoplasma-free cells were washed in Hank's Balanced Salt Solution (HBSS; Mediatech, Inc.,VA), resuspended in PBS for injection, and administered via IV tail vein injection (250 μL, 8 × 105 cells) into 40 female Nu/Nu NCR-nude mice (8 weeks of age; Charles River Laboratories, Wilmington, MA). Following administration of 32D.p210-luc+ cells, mice were then imaged 10 days later to determine baseline bioluminescence and quantify tumor-burden as previously described36. Mice were subsequently treated by oral gavage for 10 days with nilotinib (75 mg/kg qd) and reimaged (“Induction Phase” of treatment); mice were at this stage considered to have minimal residual disease, with reduced tumor burden > 2 logs. Mice were then randomly divided into four treatment groups (“Consolidation Phase” of treatment addressing minimum residual disease) with similar mean bioluminescence (n=8 or 9 per group): Group 1: Vehicle (PEG300 po), Group 2: plerixafor (5 mg/kg sq qd), Group 3: nilotinib (75mg/kg po qd), and Group 4: Combination (plerixafor+nilotinib). At the end of the study, tissues were preserved in 10% formalin. Samples, which includedvital organs, were then sent for histopathological analysis.

Histopathology

Formalin-fixed tissues were dissected and processed routinely for paraffin embedding and sectioning. Six micron sections were stained with hematoxylin and eosin.

Drug formulations for in vivo studies

Solutions of nilotinib (Novartis Pharma AG, Basel) were prepared just prior to administration, by dissolving 100 mg in 1.0 mL of NMP to give a clear solution and diluting with 9.0 mL PEG300. Plerixafor (Sigma-Aldrich, St. Louis, MO) was dissolved in water.

Statistical analysis

Results were reported as the mean ± standard deviation for three independent experiments. Samples were compared by the Student T-test, and results were considered significantly different for p values less than 0.05.

Results

CXCR4 expression in human CML cell lines

In comparison to isotype control, flow cytomery analysis showed that the human BCR-ABL-expressing cell lines, K562 and KU812F, expressed CXCR4 (Figure 1A).

Figure 1. Plerixafor decreases CML interaction with the BM microenvironment and sensitizes cells to nilotinib in vitro.

Figure 1

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The expression of CXCR4 in CML cells detected by flow cytometry (A). The effect of plerixafor (50 μM) on the adhesion of CML cells to BM microenvironment components including fibronectin (B), BMSCs (C) and endothelial cells (D). The effect of plerixafor (50 μM) on chemotaxis (E) and trans-endothelial migration (F) of CML cells in response to SDF1. The effect of plerixafor (50 μM) on resistance of CML cells to nilotinib induced by BM stroma (G).

Plerixafor reduces BCR-ABL-positive cell adhesion and migration

Direct cell interactions with BM microenvironment components have previously been shown to induce drug resistance12,13. Therefore, we tested the effect of plerixafor on the adhesion of CML cells to BM microenvironment components, such as the extracellular matrix component, fibronectin (Figure 1B), BMSCs (Figure 1C) and endothelial cells (Figure 1D). We found that plerixafor decreased the adhesion of CML cells to the BM microenvironment components. Moreover, SDF1 secreted in BM is known to induce chemotaxis of neoplastic cells to home to the protective BM. Therefore, we tested the effect of plerixafor on the chemotaxis of CML cells in response to SDF1. Figure 1E shows that SDF1 induced CML chemotaxis, which was inhibited by plerixafor. To complete the homing process, cells need to migrate through the walls of the blood vessels into the BM niche in a process termed “trans-endothelial migration,” which involves both chemotaxis and adhesion to endothelial cells. Therefore, we tested the effect of plerixafor on the transendothelial migration of CML cells. Figure 1F shows that SDF1 induced trans-endothelial migration of CML cells, which was inhibited by plerixafor.

Plerixafor reduces drug resistance of CML cells to nilotinib induced by BMSCs

To demonstrate the role of the BM stroma in drug resistance in CML, we tested the effect of nilotinib on induction of apoptosis of CML cells in presence or absence of BM stroma. Figure 1G shows that BM stroma reduced the apoptosis of CML cells induced by nilotinib. Addition of plerixafor to the co-culture system reversed the protective effect induced by the stroma, and re-sensitized the CML cells to nilotinib. The addition of plerixafor to nilotinib had no additive effect on apoptosis of CML cells when cultured alone (not in co-culture with stroma). These results suggest that disruption of the interaction of CML cells with the BM microenvironment using the CXCR4 inhibitor plerixafor sensitizes CML to nilotinib in vitro.

Plerixafor enhances tumor reduction induced by nilotinib and prolongs survival in vivo

Nilotinib was highly efficacious in reducing disease burden in leukemia-engrafted mice following 10 days of administration (Figure 2A). However, with continued treatment, animals developed resistance to nilotinib with disease burden increasing despite continued therapy (Figure 2B). Although plerixafor had no single-agent activity, combination with nilotinib significantly delayed time to relapse, and significantly prolonged survival when compared to nilotinib monotherapy (p<0.0001) (Figure 2B,C). There was no significant effect of drug treatments on the body weights of the mice (Figure 2D). Since plerixafor had no monotherapeutic efficacy, there results demonstrate that plerixafor, at a well-tolerated dose, acts synergistically with nilotinib to suppress the growth of 32D.p210 leukemia.

Figure 2. Plerixafor enhances tumor reduction induced by nilotinib and prolongs survival in vivo.

Figure 2

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(A) Tumor Burden Curve: Mice pretreated with 75 mg/kg po qd nilotinib for 10 days (“Induction Phase”). Minimal residual disease was achieved by treatment of mice with 75mg/kg po qd nilotinib for 10 days. This regimen reduced tumor burden > 2 logs. (B) Tumor Burden Curves: in vivo administration of nilotinib and plerixafor versus nilotinib only and plerixafor only (“Consolidation Phase”) in mice pretreated with 75 mg/kg po qd nilotinib for 10 days (“Induction Phase”). (C) Survival Curves: in vivo administration of nilotinib and plerixafor versus nilotinib only and plerixafor only (“Consolidation Phase”), in mice pretreated with 75 mg/kg po qd nilotinib for 10 days (“Induction Phase”). p-value (all mice): <0.0001; p-value (nilotinib vs. nilotinib+plerixafor): <0.0001; p-value (plerixafor vs. vehicle): 0.5710. (D) Mean weight of treatment groups.

The highest leukemia burden was compared in each of a pair of brain tissue slices from two vehicle-treated mice, two plerixafor only-treated mice, two nilotinib only-treated mice, and two combination-treated mice (Supplementary Figure 1). From the representative samples sent for histopathological analysis, control and treatment samples were generally comparable to one another and there was comparable focal infiltration into meninges of brain and spinal cord.

Mobilization of leukemic stem cells by plerixafor

An independent in vivo study was performed to confirm that plerixafor mobilizes leukemic stem cells into the peripheral blood of mice as its primary mechanism of action. Mice were treated for three days with either vehicle control (PEG300, po qd) or plerixafor (5 mg/kg sq qd) (Figure 3). Luciferase activity was quantified in peripheral blood samples taken from mice on days 0 and 3 and compared between vehicle control mice and plerixafor-treated mice. The level of luciferase activity, which is a measure of the quantity of luciferase-expressing 32D.p210 cells, in the peripheral blood of plerixafor-treated mice on day 3 was significantly higher than day 0 (p=0.03). In contrast, the level of luciferase activity in the peripheral blood of vehicle control-treated mice was comparable between days 0 and 3. This suggests mobilization of 32D.p210-luc+ cells from the bone marrow microenvironment into the peripheral blood by plerixafor.

Figure 3. Mobilization of leukemic stem cells by plerixafor.

Figure 3

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Discussion

CXCR4 antagonists have demonstrated ability to enhance the efficacy of chemotherapeutic agents and tyrosine kinase inhibitors by disrupting the communication of malignant cells with their surrounding BM microenvironment, mobilizing these cells into the circulation, and thus rendering the cells more susceptible to the inhibitory effects of therapeutic drugs. Indeed, in vitro and in vivo studies have shown plerixafor, an antagonist/partial agonist of the alpha-chemokine receptor CXCR4 and allosteric agonist of CXCR731, to be effective in mobilizing hematopoietic cells from BM and enhancing chemotherapy- or tyrosine kinase inhibitor-induced apoptosis of bone marrow stroma-protected acute myeloid leukemia (AML)32, 37, 38, multiple myeloma12, and CML28, 39.

For CML, studies demonstrating the ability of plerixafor to reduce tumor burden in vivo and prolong lifespan following treatment with tyrosine kinase inhibitors have been lacking. To demonstrate potentiation of tyrosine kinase activity in CML by plerixafor, Vianello et al. used measurement of the percentages of human CD19+ cells that engrafted in the BM and spleen of mice following co-cultivation of imatinib-treated BCR-ABL-expressing cells with mesenchymal stromal cells and pre-treatment with plerixafor. They found significant reductions in engraftment for cells pretreated with plerixafor and then treated with imatinib, suggesting a restoration of the susceptibility of the cells to the ABL inhibitor by plerixafor. Thus, to date, there has been no full report of an investigation of the long-term effects of combined treatment with plerixafor and an ABL inhibitor on leukemic burden and overall survival in mice harboring BCR-ABL-positive leukemia.

Our goal in this study was to see whether pharmacological targeting of drug resistance mechanisms synergizes with existing, targeted therapies as a way to enhance clinical efficacy by eliminating, or delaying the onset of, residual disease. To accomplish this, we employed a functional in vivo assay system that allows monitoring of the growth of progressive disease, as well as baseline level (“residual” disease) resulting from treatment with a moderate-to-high dose of nilotinib. This model has been successfully used previously to demonstrate synergy between the IAP inhibitor, LCL161, and nilotinib that delayed the onset of residual disease following treatment of mice with a moderate-to-high dose of nilotinib40. Here, we demonstrated the ability of stem cell mobilization to enhance the efficacy of nilotinib by suppressing leukemia recurrence following nilotinib treatment.

Consistent with the ability of plerixafor to mobilize leukemic stem cells via detachment from the BM microenvironment into the peripheral blood of mice, plerixafor diminished BCR-ABL-positive cell migration and reduced adhesion of these cells to fibronectin, BMSCs and HUVECs in vitro. Stroma reduced the apoptosis of CML cells induced by nilotinib. Addition of plerixafor to the co-culture system reversed the protective effect of the stroma and re-sensitized the CML cells to nilotinib. These results suggest that disruption of the interaction of CML cells with the BM microenvironment using the CXCR4 inhibitor plerixafor sensitizes CML to nilotinib in vitro.

A previous report41 tested the combination of plerixafor with tyrosine kinase inhibitors imatinib and dasatinib, respectively, in a murine CML retroviral transduction/transplantation model in which minimal residual disease was not achieved despite inhibition of BCR-ABL activity. In the model utilized in this report, which mimics highly active CML, the combination of plerixafor with tyrosine kinase inhibitors did not reduce leukemia burden and instead promoted extra medullary leukemic infiltrates in vital organs including the brain. There are several differences between the murine model used in this report and that used herein that may account for the disparity in results achieved. First, we found no evidence to suggest that the combination of plerixafor with BCR-ABL inhibition in our mouse model led to extramedullary infiltration of leukemic cells. In particular, with regard to the lack of brain-infiltration, the CML model used in our study does not involve the irradiation of animals. This procedure might have damaged the blood-brain barrier in the bone-marrow transplant model employed by Agarwal et al. Indeed, another study42 confirmed that in the absence of irradiation, plerixafor does not lead to brain infiltration of bone marrow-derived cells. In the present study and in contrast to Agarwal et al., nilotinib achieves minimal residual disease, such that an effect of plerixafor on stem cells is evident. In the absence of irradiation, toxicity resulting from extramedullary infiltration of leukemic cells was not observed. In the Agarwal et al. report, imatinibhas a poor PK in mice and despite being administered at 100 mg/kg BID, there was a persistence of leukemic cells in the blood and little margin to show cooperativity relating to stem cell moblization due to this and the fact that plerixafor was likely causing toxicity due to extramedullary infiltration because of the irradiation. Finally, in the Agarwal et al. report, dasatinib, which has high potency but a very short plasma half-life that lowers its overall efficacy, was tested in combination with plerixafor not against minimal residual disease, but when there existed a significant burden of circulating leukemic cells. When plerixafor was administered, leukemic cells migrated into vital organs, leading to enhanced toxicity.

In conclusion, we demonstrated the ability of plerixafor to delay the onset of recurring BCR-ABL- positive disease in mice carrying an extremely low tumor burden following treatment with a moderate-to-high dose of nilotinib. These results support the idea of using stem cell mobilization in conjunction with targeted tyrosine kinase inhibition to override drug resistance and suppress or eradicate residual disease.

Supplementary Material

Figure S1

Supplementary Figure 1. Histopathology: brain tissue sections derived from two representative mice per treatment group. Green arrows depict staining positive for the presence of tumor, whereas those sections with an absence of arrows showed no leukemia infiltration.

Acknowledgments

Grant Support: J.D.G is supported by NIH grant CA66996.

Footnotes

J.D.G. has a financial interest with Novartis Pharma AG. J.D.G. and A.L.K. have a financial interest with Novartis Pharma AG. PWM is an employee of Novartis. None of the authors included in this manuscript have a financial conflict of interest.

References

  • 1.Mahon FX, Rea D, Guilhot J, Guilhot F, Huguet F, Nicolini F, et al. Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 yeras: the prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol. 2010;11:1029–35. doi: 10.1016/S1470-2045(10)70233-3. [DOI] [PubMed] [Google Scholar]
  • 2.Chomel JC, Bonnet ML, Sorel N, Bertrand A, Meunier MC, Fichelson S, et al. Leukemic stem cell persistence in chronic myeloid leukemia patients with sustained undetectable molecular residual disease. Blood. 2011;118:3657–3660. doi: 10.1182/blood-2011-02-335497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Konig H, Holtz M, Modi H, Manley P, Holyoake TL, Forman SJ, et al. Enhanced BCR-ABL kinase inhibition does not result in increased inhibition of downstream signaling pathways or increased growth suppression in CML progenitors. Leukemia. 2008;22:748–55. doi: 10.1038/sj.leu.2405086. [DOI] [PubMed] [Google Scholar]
  • 4.Kumagai M, Manabe A, Pui CH, Behm FG, Raimondi SC, Hancock ML, et al. Stroma-supported culture in childhood B-lineage acute lymphoblastic leukemia cells predicts treatment outcome. J Clin Invest. 1996;97:755–760. doi: 10.1172/JCI118474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Ashley DM, Bol SJ, Kannourakis G. Human bone marrow stromal cell contact and soluble factors have different effects on the survival and proliferation of paediatric B-lineage acute lymphoblastic leukaemic blasts. Leuk Res. 1994;18:337–346. doi: 10.1016/0145-2126(94)90017-5. [DOI] [PubMed] [Google Scholar]
  • 6.Bradstock K, Bianchi A, Makrynikola V, Filshie R, Gottlieb D. Long-term survival and proliferation of precursor B acute lymphoblastic leukemia cells on human bone marrow stroma. Leukemia. 1996;10:813–820. [PubMed] [Google Scholar]
  • 7.Rafii S, Mohle R, Shapiro F, Frey BM, Moore MA. Regulation of hematopoiesis by microvascular endothelium. Leuk Lymphoma. 1997;27:375–386. doi: 10.3109/10428199709058305. [DOI] [PubMed] [Google Scholar]
  • 8.Lagneaux L, Delforge A, Bron D, De Bruyn C, Stryckmans P. Chronic lymphocytic leukemic B cells but not normal B cells are rescued from apoptosis by contact with normal bone marrow stromal cells. Blood. 1998;91:2387–2396. [PubMed] [Google Scholar]
  • 9.Lagneaux L, Delforge A, De Bruyn C, Bernier M, Bron D. Adhesion to bone marrow stroma inhibits apoptosis of chronic lymphocytic leukemia cells. Leukemia Lymphoma. 1999;35:445–453. doi: 10.1080/10428199909169609. [DOI] [PubMed] [Google Scholar]
  • 10.Konopleva M, Konoplev S, Hu W, Zaritskey AY, Afanasiev BV, Andreeff M. Stromal cells prevent apoptosis of AML cells by up-regulation of anti-apoptotic proteins. Leukemia. 2002;16:1713–1724. doi: 10.1038/sj.leu.2402608. [DOI] [PubMed] [Google Scholar]
  • 11.Litwin C, Leong KG, Zapf R, Sutherland H, Naiman SC, Karsan A. Role of the microenvironment in promoting angiogenesis in acute myeloid leukemia. Am J Hematol. 2002;70:22–30. doi: 10.1002/ajh.10092. [DOI] [PubMed] [Google Scholar]
  • 12.Azab AK, Runnels JM, Pitsillides C, Moreau AS, Azab F, Leleu X, et al. CXCR4 inhibitor AMD3100 disrupts the interaction of multiple myeloma cells with the bone marrow microenvironment and enhances their sensitivity to therapy. Blood. 2009a;113:4341–51. doi: 10.1182/blood-2008-10-186668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Azab AK, Azab F, Blotta S, Pitsillides CM, Thompson B, Runnels JM, et al. RhoA and Rac1 GTPases play major and differential roles in stromal cell-derived factor-1-induced cell adhesion and chemotaxis in multiple myeloma. Blood. 2009b;114:619–29. doi: 10.1182/blood-2009-01-199281. IF=10.5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Azab AK, Azab F, Maiso P, Quang P, Roccaro AM, Sacco A, et al. FGFR3 is overexpressed in Waldenström macroglobulinemia and its inhibition by Dovitinib induces apoptosis, and overcomes stroma-induced proliferation. Clin Cancer Res. 2011;17:4389–4399. doi: 10.1158/1078-0432.CCR-10-2772. [DOI] [PubMed] [Google Scholar]
  • 15.Azab F, Azab AK, Maiso P, Quang P, Roccaro AM, Sacco A, et al. Eph-B2/ephrin-B2 interaction plays a major role in the adhesion and survival of WM cells in the context of the bone marrow microenvironment. Eph-B2/ephrin-B2 interaction plays a major role in the adhesion and survival of Waldenstrom Macroglobulinemia. Clin Cancer Res. 2011b doi: 10.1158/1078-0432. [DOI] [Google Scholar]
  • 16.Azab AK, Quang P, Azab F, Pitsillides C, Tompson B, Chonghaile T, et al. P-selectin glycoprotein ligand regulates the interaction of multiple myeloma cells with the bone marrow microenvironment. Blood. 2011c doi: 10.1182/blood-2011-07-368050. In press. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Verfaillie CM. Soluble factor(s) produced by human bone marrow stroma increase cytokine-induced proliferation and maturation of primitive hematopoietic progenitors while preventing their terminal differentiation. Blood. 1993;82:2045–53. [PubMed] [Google Scholar]
  • 18.Liesveld JL, Harbol AW, Abboud CN. Stem cell factor and stromal cell co-culture prevent apoptosis in a subculture of the megakaryoblastic cell line, UT-7. Leuk Res. 1996;20:591–600. doi: 10.1016/0145-2126(95)00171-9. [DOI] [PubMed] [Google Scholar]
  • 19.Harrison PR, Nibbs RJ, Bartholomew C, O'Prey J, Qiu J, Walker M, et al. Molecular mechanisms involved in long-term maintenance of erythroleukaemia cells by stromal cells. Leukemia. 1997;11:474–477. [PubMed] [Google Scholar]
  • 20.Breems DA, Blokland EA, Ploemacher RE. Stroma-conditioned media improve expansion of human primitive hematopoietic stem cells and progenitor cells. Leukemia. 1997;11:42–50. doi: 10.1038/sj.leu.2400530. [DOI] [PubMed] [Google Scholar]
  • 21.O'Prey J, Leslie N, Itoh K, Ostertag W, Bartholomew C, Harrison PR. Both stroma and stem cell factor maintain long-term growth of ELM erythroleukemia cells, but only stroma prevents erythroid differentiation in response to erythropoietin and interleukin-3. Blood. 1998;91:1548–1555. [PubMed] [Google Scholar]
  • 22.Leslie NR, O'Prey J, Bartholomew C, Harrison PR. An activating mutation in the kit receptor abolishes the stroma requirement for growth of ELM erythroleukemia cells, but does not prevent their differentiation in response to erythropoietin. Blood. 1998;92:4798–4807. [PubMed] [Google Scholar]
  • 23.Shih CC, Hu MC, Hu J, Medeiros J, Forman SJ. Long-term ex vivo maintenance and expansion of transplantable human hematopoietic stem cells. Blood. 1999;94:623–36. [PubMed] [Google Scholar]
  • 24.Shaked Y, Cervi D, Neuman M, Chen L, Klement G, Michaud CR, et al. The splenic microenvironment is a source of proangiogenesis/inflammatory mediators accelerating the expansion of murine erythroleukemic cells. Blood. 2005;105:4500–4507. doi: 10.1182/blood-2004-08-3210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Despars G, O'Neill HC. Splenic endothelial cell lines support development of dendritic cells from bone marrow. Stem Cells. 2006;24:1496–1504. doi: 10.1634/stemcells.2005-0530. [DOI] [PubMed] [Google Scholar]
  • 26.Weisberg E, Wright RD, McMillin DW, Mitsiades C, Ray A, Barrett R, et al. Stromal-mediated protection of tyrosine kinase inhibitor-treated BCR-ABL-expressing leukemia cells. Mol Cancer Ther. 2008;7:1121–9. doi: 10.1158/1535-7163.MCT-07-2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Jin L, Tabe Y, Konoplev S, Xu Y, Levsath CE, Lu H, et al. CXCR4 up-regulation by imatinib induces chronic myelogenous leukemia (CML) cell migration to bone marrow stroma and promotes survival of quiescent CML cells. Mol Cancer Ther. 2008;7:48–58. doi: 10.1158/1535-7163.MCT-07-0042. [DOI] [PubMed] [Google Scholar]
  • 28.Dillmann F, Veldwijk MR, Laufs S, Sperandio M, Calandra G, Wenz F, et al. Plerixafor inhibits chemotaxis toward SDF-1 and CXCR4-mediated stroma contact in a dose-dependent manner resulting in increased susceptibility of BCR-ABL+ cell to Imatinib and Nilotinib. Leuk Lymphoma. 2009;50:1676–86. doi: 10.1080/10428190903150847. [DOI] [PubMed] [Google Scholar]
  • 29.Geay JF, Buet D, Zhang Y, Foudi A, Jarrier P, Berthebaud M, et al. p210BCR-ABL inhibits SDF-1 chemotactic response via alteration of CXCR4 signaling and down-regulation of CXCR4 expression. Cancer Res. 2005;65:2676–83. doi: 10.1158/0008-5472.CAN-04-2152. [DOI] [PubMed] [Google Scholar]
  • 30.Zeng Z, Samudio IJ, Munsell M, An J, Huang Z, Estey E, et al. Inhibition of CXCR4 with the novel RCP168 peptide overcomes stroma-mediated chemoresistance in chronic and acute leukemias. Mol Cancer Ther. 2006;5:3113–21. doi: 10.1158/1535-7163.MCT-06-0228. [DOI] [PubMed] [Google Scholar]
  • 31.Kalatskaya I, Berchiche Y, Gravel S, Limberg B, Rosenbaum J, Heveker N. AMD3100 is a CXCR7 ligand with allosteric agonist properties. Molecular Pharmocology. 2009;75:1240–1247. doi: 10.1124/mol.108.053389. [DOI] [PubMed] [Google Scholar]
  • 32.Nervi B, Ramirez P, Rettig MP, Uy GL, Holt MS, Ritchey JK, et al. Chemosensitization of acute myeloid leukemia (AML) following mobilization by the CXCR4 antagonist AMD3100. Blood. 2009;113:6206–14. doi: 10.1182/blood-2008-06-162123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Alsayed Y, Ngo H, Runnels J, Leleu X, Singha UK, Pitsillides CM, et al. Mechanisms of regulation of CXCR4/SDF1 (CXCL12)-dependent migration and homing in multiple myeloma. Blood. 2007;109:2708–2717. doi: 10.1182/blood-2006-07-035857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Matulonis U, Salgia R, Okuda K, Druker B, Griffin JD. IL-3 and p210 BCR-ABL activate both unique and overlapping pathways of signal transduction in a factor-dependent myeloid cell line. Exp Hematol. 1993;21:1460–1466. [PubMed] [Google Scholar]
  • 35.Weisberg E, Manley PW, Breitenstein W, Brüggen J, Cowan-Jacob SW, Ray A, et al. Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl. Cancer Cell. 2005;7:129–41. doi: 10.1016/j.ccr.2005.01.007. [DOI] [PubMed] [Google Scholar]
  • 36.Armstrong SA, Kung AL, Mabon ME, Silverman LB, Stam RW, Den Boer ML, et al. Validation of a therapeutic target identified by gene expression based classification. Cancer Cell. 2003;3:173–83. doi: 10.1016/s1535-6108(03)00003-5. [DOI] [PubMed] [Google Scholar]
  • 37.Jacobi A, Thieme S, Lehmann R, Ugarte F, Malech HL, Koch S, et al. Impact of CXCR4 inhibition on FLT3-ITD-positive human AML blasts. Exp Hematol. 2010;38:180–90. doi: 10.1016/j.exphem.2009.12.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Liesveld JL, Bechelli J, Rosell K, Lu C, Bridger G, Phillips G, 2nd, et al. Effects of AMD3100 on transmigration and survival of acute myelogenous leukemia cells. Leuk Res. 2007;31:1553–63. doi: 10.1016/j.leukres.2007.02.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Vianello F, Villanova F, Tisato V, Lymperi S, Ho KK, Gomes AR, et al. Bone marrow mesenchymal stromal cells non-selectively protect chronic myeloid leukemia cells from imatinib-induced apoptosis via the CXCR4/CXCL12 axis. Haematologica. 2010;95:1081–1089. doi: 10.3324/haematol.2009.017178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Weisberg E, Ray A, Barrett R, Nelson E, Christie AL, Porter D, et al. Smac mimetics: implications for enhancement of targeted therapies in leukemia. Leukemia. 2010;24:2100–9. doi: 10.1038/leu.2010.212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Agarwal A, Fleischman AG, Petersen CL, Loriaux MM, Woltjer R, Druker BJ, et al. Effects of Plerixafor (AMD3100) in Combination with Tyrosine Kinase Inhibition in a Murine Mouse Model of CML. 52nd ASH Annual Meeting and Exposition; Dec 4-7, 2010. Abstract #3389. [Google Scholar]
  • 42.Burrell K. Role of bone marrow derived cells in response to ionizing radiation in normal brain. Abstract #566. AACR. 2011 [Google Scholar]

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Supplementary Materials

Figure S1

Supplementary Figure 1. Histopathology: brain tissue sections derived from two representative mice per treatment group. Green arrows depict staining positive for the presence of tumor, whereas those sections with an absence of arrows showed no leukemia infiltration.

NIHMS582723-supplement-Figure_S1.jpg (125.4KB, jpg)

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