CXCR4 Inhibition as a Therapeutic Strategy in Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 456-456
Author(s):  
Zhihong Zeng ◽  
Randall L. Evans ◽  
Ziwei Huang ◽  
Michael Andreeff ◽  
Marina Konopleva
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stromal Cells ◽  
Surface Expression ◽  
Bone Marrow Microenvironment ◽  
Jurkat Cells ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Viable Cells ◽  
Induced Apoptosis

Abstract The chemokine receptor CXCR4 is critically involved in the migration of hematopoietic cells to the stroma derived factor (SDF-1α)-producing bone marrow microenvironment. We and others have previously demonstrated that stromal-leukemic interactions mediate protection of leukemic cells from chemotherapy-induced apoptosis. (Konopleva et al, Leukemia 2002; Tabe, Konopleva, et al, Blood 2004; Burger JA et al., Blood 2000). Using peptide based CXCR4 inhibitors, derived from the chemokine viral macrophage inflammatory protein II (vMIP II), we tested the hypothesis that CXCR4 inhibition interferes with stromal/leukemia cell interactions resulting in increased sensitivity to chemotherapy. CXCR4 was highly expressed on the cell surface of CML myeloid blood crisis cells (KBM5), KBM5/STI-resistant cells, lymphoid CEM and Jurkat cells, myeloid leukemic OCI-AML3 and U937 cells. In contrast, NB4 and TF-1 cells expressed low-levels surface CXCR4, and no surface expression was detected on KG-1 and HL-60 leukemic cells. Among CXCR4(+) cell lines, Jurkat cells demonstrated the highest chemoattractive response to SDF-1α(23 +/− 0.03% migration at SDF-1α50ng/ml, and 54 +/− 0.01% at 100ng/ml). The ability of three CXCR4 inhibitors to inhibit chemotaxis of Jurkat cells was examined in a standard migration assay. Results indicate that D10-vMIP-II, a polypeptide with the first 10 amino acids substituted by the D isoform, exhibits the strongest antagonistic effect on SDF-1α or stromal cell induced chemoattraction. D10-vMIP-II also decreases CXCR4 surface expression in a concentration-dependent manner: flow cytometry and live cell confocal microscopy revealed that within 30min of exposure D10-vMIP-II causes CXCR4 internalization that persisted for at least 4 hrs at 0.01μM and for 24 hrs at 0.1μM. Analysis of SDF-1α-mediated signaling demonstrated that D10-vMIP-II inhibits AKT and ERK phosphorylation. Finally, we examined the effects of D10-vMIP-II on the response to chemotherapy of leukemic cells co-cultured with MS5 stromal cells. Pre-treatment of Jurkat cells enhanced doxorubicin-induced apoptosis: Doxorubicin alone (10μM) 75 +/− 0.07% viable cells compared to control; Doxorubicin and D10-vMIP-II: 53 +/− 0.04% viable cells. Furthermore, D10-vMIP-II enhanced the sensitivity of primary CLL cells to Fludarabine in the in vitro stromal co-culture system. CLL samples with high surface expression of CXCR4 (n=3) co-cultured with stromal MS-5 cells were pre-treated with 0.1μM D10-vMIP II followed by 10μM Fludarabine (9-β-D-arabinofuranosyl-2-fluoroadenine). Stromal cells prevented Fludarabine-induced killing (64%±16.2 viable cells in stromal co-culture compared to 31% viable cells in medium only). Inhibition of CXCR4 signaling abrogated this protective effect and diminished CLL cell survival (26.9±7.1% viable cells, p=0.03 compared to Fludarabine-treated CLL cells co-cultured with MS-5). This growth inhibition was mediated by apoptosis induction as determined by CD45/annexinV flow cytometry (DMSO, 14.49±5.3% annexinV(+) leukemic cells; Fludarabine, 47.2±24.9%; D10-vMIP II followed by Fludarabine, 61.3±18.9%). Taken together, our data suggest that SDF-1α/CXCR4 interactions contribute to the resistance of leukemic cells to chemotherapy-induced apoptosis. Disruption of these interactions by the potent CXCR4 inhibitor D10-vMIP-II represents a novel strategy for the targeting leukemic cells within their bone marrow microenvironment.

Disruption of Leukemia/Stroma Cell Interactions by CXCR4 Antagonist AMD3465 Enhances Chemotherapy-Induced Apoptosis in AML.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 474-474 ◽  
Author(s):  
Zhihong Zeng ◽  
Marina Konopleva ◽  
Billie J. Nowak ◽  
William Plunkett ◽  
Gautam Borthakur ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cells ◽  
Leukemia Cell ◽  
Surface Expression ◽  
Bone Marrow Microenvironment ◽  
Cell Interactions ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Induced Apoptosis

Abstract Chemokine receptor CXCR4 is critically involved in the migration of hematopoietic cells to the stromal derived factor (SDF-1α)-producing bone marrow microenvironment. We and others have previously demonstrated that stroma/leukemia interactions mediate protection of leukemic cells from chemotherapy-induced apoptosis (Konopleva, Leukemia2002:1713; Burger Blood2000: 2655). Using AMD3465, the second-generation small-molecule CXCR4 inhibitor with a greater potency than AMD3100, we tested the hypothesis that CXCR4 inhibition interferes with stromal/leukemia cell interactions resulting in increased sensitivity to chemotherapy. Our results showed that AMD3465 inhibited surface expression of CXCR4 on AML cell lines in a dose dependent manner. AMD3465 (1μM) significantly inhibited SDF-1α and stromal (MS-5)-induced migration of OCI-AML2 cells (78% and 54% inhibition, respectively), U937 cells (71% and 41.3%) and diminished SDF-1α- or stromal-induced migration of leukemic blasts from four primary AML samples tested (SDF-1α, 43.4 ± 8.6%, MS-5, 38.4 ± 8.5% inhibition). In in vitro co-culture systems, stromal cells significantly protected leukemic cell lines and primary AML cells from spontaneous and chemotherapy induced apoptosis (p<0.01; p<0.001). Measurements of intracellular Ara-CTP levels determined by HPLC showed that stromal cells diminished incorporation of Ara-C into leukemic cells by 20%. AMD3465 enhanced AraC- and Busulfan-induced apoptosis by 44% and 69%, respectively. Western blot revealed that AMD3465 downregulated AKT signaling in AML cells. Most importantly, it decreased stroma-mediated protection from AraC-induced apoptosis in five out of ten primary AML samples with surface expression of functional CXCR4 (mean increase, 29.9±19.5% compared to chemotherapy alone). Curiously, the highest sensitization was observed in a sample from AML patient harboring Flt3/ITD mutation (Ara-C, 30.3% annexinV(+); Ara-C+AMD, 62.8%), confirming recently documented role for Flt3/ITD in modulation of CXCR4 signaling (Fukuda, Blood2005:3117). Taken together, our data suggest that SDF-1α/ CXCR4 interactions contribute to the resistance of leukemic cells to chemotherapy-induced apoptosis. Disruption of these interactions by the potent CXCR4 inhibitor AMD3465 represents a novel strategy for targeting leukemia cell/bone marrow microenvironment interactions. A clinical trial testing this concept in patients with AML is in preparation.

Disruption of Leukemia/Stroma Cell Interactions by CXCR4 Antagonist CTCE-9908 Enhances Chemotherapy-Induced Apoptosis in AML

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2415-2415
Author(s):  
Hongbo Lu ◽  
Zhihong Zeng ◽  
Yuexi Shi ◽  
Sergej Konoplev ◽  
Donald Wong ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Leukemia Cell ◽  
Bone Marrow Microenvironment ◽  
Cell Interactions ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Induced Apoptosis ◽  
Dose Dependent

Abstract The chemokine receptor CXCR4 is critically involved in the migration of hematopoietic cells towards the stromal derived factor (SDF-1α)-producing bone marrow microenvironment. We and others have previously demonstrated that stroma/leukemia interactions mediate protection of leukemic cells from chemotherapy-induced apoptosis (Konopleva, Leukemia 2002). Using a peptide analog of SDF-1α designated CTCE-9908, we tested the hypothesis that CXCR4 inhibition interferes with stromal/leukemia cell interactions resulting in increased sensitivity to chemotherapy. Our results showed that CTCE-9908 significantly inhibits SDF-1α-induced migration of U937 (43% inhibition) and OCI-AML3 cells (40% inhibition) in a dose-dependent manner. In three of the four primary AML samples which expressed CXCR4 on cell surface and migrated in response to SDF-1α, 50 μg/ml CTCE-9908 reduced SDF-1α-induced migration of leukemic blasts (60%, 19% and 50% inhibition respectively). In in vitro co-culture systems, stromal cells significantly protected OCI-AML3 cells from chemotherapy induced apoptosis [no MS-5, 75.2±5.2% annexinV(+); with MS-5, 59±1.1% annexinV(+)]. Western blot analysis revealed that CTCE-9908 inhibits Akt and Erk phosphorylation in a dose-dependent manner in the OCI-AML3 cell line stimulated by SDF-1α. Blockade of CXCR4 expression with CTCE-9908 markedly abrogated the protective effects of stromal cells on OCI-AML3 [Ara-C, 59±1.1% annexinV(+); Ara-C + CTCE-9908, 76.9±1.35 annexinV(+)]. Most importantly, it decreased stroma-mediated protection from AraC-induced apoptosis in four out of five primary AML samples with surface expression of functional CXCR4 (mean increase, 25.1±9.3% compared to chemotherapy alone). In vivo, subcutaneous administration of 1.25mg CTCE-9908 induced mobilization of leukemic cells from primary AML patient transplanted into NOD/Scid-IL2Rγ-KO mice (from 15% to 27% circulating leukemic cells 1 hour post CTCE-9908 injection). Taken together, our data suggest that SDF-1α/CXCR4 interactions contribute to the resistance of leukemic cells to chemotherapy-induced apoptosis via retention of leukemic cells in the bone marrow microenvironment niches. Disruption of these interactions by the potent CXCR4 inhibitor CTCE-9908 represents a novel strategy for targeting leukemia cell/bone marrow microenvironment interaction. Based on these observations, in vivo experiments are ongoing to characterize the efficacy of chemotherapy combined with CTCE-9908.

Massive Mobilization of AML Cells into Circulation by Disruption of Leukemia/Stroma Cell Interactions Using CXCR4 Antagonist AMD3100: First Evidence in Patients and Potential for Abolishing Bone Marrow Microenvironment-Mediated Resistance.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 568-568 ◽  
Author(s):  
Michael Andreeff ◽  
Sergej Konoplev ◽  
Rui-Yu Wang ◽  
Zhihong Zeng ◽  
Teresa McQueen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Leukemia Cell ◽  
Surface Expression ◽  
Bone Marrow Microenvironment ◽  
Leukemic Cells ◽  
Fish Analysis ◽  
Cxcr4 Antagonist ◽  
Induced Apoptosis

Abstract The chemokine receptor CXCR4 is critically involved in migration of hematopoietic cells to the stromal derived factor (SDF-1α)-producing bone marrow microenvironment. CXCR4 is regulated in part by mutant FLT3 signaling, but in a series of 122 AML samples with diploid karyotype and lack of FLT3 mutation (ITD), high CXCR4 expression negatively correlated with DFS and OS (p=0.03 and p=0.04, respectively), after multivariate analysis (Konoplev, ASH 2006). We hypothesized that inhibition of SDF-1α-/CXCR4 interactions would result in mobilization of leukemic blasts from the bone marrow into circulation. The in vivo effect of the CXCR4 antagonist AMD3100 was studied in three patients with AML, who had insufficient mobilization of CD34+ cells for autologous stem cell transplantation with G-CSF and/or cytoxan. The combination of G-CSF (10 μg/kg QD) and AMD3100 (240 μg/kg QD SC starting on d4 and repeated for 3–4 days) resulted in massive mobilization of leukemic cells into the circulation in a time-dependent fashion, as determined by flow cytometry and interphase FISH analysis of their respective cytogenetic abnormalities. Patient # Cytogenetics % (+) cells % (+) cells Apheresis FCM Day 2 Day 4/5 CD34x106/kg 1 Trisomy 21 22.6 57.0 FCM CD7/33 22.0 2 Trisomy 9 28.6 68.6 Inv 16 29.0 75.8 4.8 FCM CD13/33 74.0 3 Mono 17 40.4 53.4 5q31 37.5 49.6 8.7 FCM CD13/33 50.0 We and others have previously demonstrated that stroma/leukemia interactions mediate protection of leukemic cells from chemotherapy-induced apoptosis (Konopleva et al, Leukemia2002:1713). We then tested the hypothesis that CXCR4 inhibition would result in increased sensitivity to chemotherapy, using AMD3465, the second generation small-molecule CXCR4 inhibitor with greater potency than AMD3100. Results demonstrate inhibition of surface expression of CXCR4 and of SDF-1α-, and stroma(MS-5)-induced migration of AML cells. In vitro co-culture systems with stromal cells significantly protected leukemic cells (p < 0.01), while AMD3465 decreased stroma-mediated protection from AraC and Busulfan apoptosis and downregulated AKT signaling in AML cells. In a murine model of luciferase labeled Baf-FLT3ITD leukemias, AMD3465 induced massive dissemination of leukemia, which was abrogated by treatment with Sorafenib, a potent FLT3ITD inhibitor (Zhang, ASH 2006). Taken together, our data suggest that SDF-1α/CXCR4 interactions contribute to the resistance of leukemic cells to chemotherapy-induced apoptosis. Disruption of these interactions by CXCR4 inhibition results in leukemia dissemination and chemosensitization. Our results in leukemia patients provide first in man proof-of principle for a novel strategy of targeting the leukemia cell/bone marrow microenvironment interactions. A clinical trial testing this concept in patients with AML is under development.

scholarly journals Targeting the leukemia microenvironment by CXCR4 inhibition overcomes resistance to kinase inhibitors and chemotherapy in AML

Blood ◽  
2009 ◽  
Vol 113 (24) ◽  
pp. 6215-6224 ◽  
Author(s):  
Zhihong Zeng ◽  
Yue Xi Shi ◽  
Ismael J. Samudio ◽  
Rui-Yu Wang ◽  
Xiaoyang Ling ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Fetal Liver ◽  
Bone Marrow Microenvironment ◽  
Leukemic Cells ◽  
In Vivo Studies ◽  
Protective Effects ◽  
Cxcr4 Signaling ◽  
Induced Apoptosis

Abstract SDF-1α/CXCR4 signaling plays a key role in leukemia/bone marrow microenvironment interactions. We previously reported that bone marrow–derived stromal cells inhibit chemotherapy-induced apoptosis in acute myeloid leukemia (AML). Here we demonstrate that the CXCR4 inhibitor AMD3465 antagonized stromal-derived factor 1α (SDF-1α)–induced and stroma-induced chemotaxis and inhibited SDF-1α–induced activation of prosurvival signaling pathways in leukemic cells. Further, CXCR4 inhibition partially abrogated the protective effects of stromal cells on chemotherapy-induced apoptosis in AML cells. Fetal liver tyrosine kinase-3 (FLT3) gene mutations activate CXCR4 signaling, and coculture with stromal cells significantly diminished antileukemia effects of FLT3 inhibitors in cells with mutated FLT3. Notably, CXCR4 inhibition increased the sensitivity of FLT3-mutated leukemic cells to the apoptogenic effects of the FLT3 inhibitor sorafenib. In vivo studies demonstrated that AMD3465, alone or in combination with granulocyte colony-stimulating factor, induced mobilization of AML cells and progenitor cells into circulation and enhanced antileukemic effects of chemotherapy and sorafenib, resulting in markedly reduced leukemia burden and prolonged survival of the animals. These findings indicate that SDF-1α/CXCR4 interactions contribute to the resistance of leukemic cells to signal transduction inhibitor– and chemotherapy-induced apoptosis in systems mimicking the physiologic microenvironment. Disruption of these interactions with CXCR4 inhibitors represents a novel strategy of sensitizing leukemic cells by targeting their protective bone marrow microenvironment.

Differential Expression of Adhesion Molecule Receptors May Influence Bone Marrow Microenvironment-Mediated Protection of Leukemia-Initiating Cells (LICs) in Infant MLL-rearranged (MLL-R) Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1585-1585
Author(s):  
Edward Allan R. Sison ◽  
Amos S. Gaikwad ◽  
Gaye Jenkins ◽  
Terzah M. Horton ◽  
Patrick Brown ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Adhesion Molecule ◽  
Stromal Cells ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Surface Expression ◽  
Vehicle Control ◽  
Spontaneous Apoptosis ◽  
Induced Apoptosis

Abstract Background: Compared to older children with ALL who achieve a minimal residual disease (MRD)-negative remission, infants with ALL who achieve an MRD-negative remission have a significantly higher rate of relapse. This suggests that, despite the achievement of MRD negativity, a very small amount of leukemia cells persist and contribute to disease relapse in infant ALL; LICs are likely to represent this persistent population of cells. Previously, we demonstrated that infant MLL-R ALL derive significantly more protection from co-culture with bone marrow (BM) stromal cells than non-MLL-R ALL. Therefore, protection by the BM microenvironment may contribute to LIC persistence in infant MLL-R ALL. We hypothesized that infant MLL-R ALL LICs have increased interactions with the BM microenvironment, through increased surface expression of adhesion molecule receptors. We also hypothesized that co-culture with normal BM stroma would protect LICs from spontaneous and chemotherapy-induced apoptosis, and AMD3100 (plerixafor) would decrease stromal protection through inhibition of CXCR4-CXCL12 signaling. Methods/Results: We analyzed 9 viably cryopreserved diagnostic samples collected from infants with MLL-R ALL (n=4 MLL-AF4, n=5 MLL-ENL). Using flow cytometry, we identified 2 phenotypically-defined LIC subpopulations, CD34+CD38+CD19+ and CD34-CD19+ (Aoki et al 2015), within the bulk leukemic blast population (CD45+). Consistent with previous findings, 34+38+19+ LICs were predominant in MLL-AF4 samples, while 34-19+ LICs were predominant in MLL-ENLsamples. Next, we measured surface expression of the adhesion molecule receptors CXCR4, CD49d (VLA-4), CXCR7, and CXCR3 (quantified as mean fluorescence intensity, MFI). Overall, surface expression of CXCR4, CD49d, CXCR7, and CXCR3 was higher in the 34+38+19+ LICs, compared to 34-19+ LICs (e.g., CXCR4 MFI 142 in 34+38+19+ vs. 76 in 34-19+, p=0.02). Next, samples were treated with dose ranges (0-30 µM) of AraC or etoposide (Etop) and cultured for 48 hours in 3 conditions: 1) off stroma, 2) on stroma, or 3) on stroma with the CXCR4 inhibitor AMD3100 (10 μM). Stromal cells were cultured from a healthy BM donor. In vehicle control-treated 34+38+19+ LICs, stromal co-culture led to increased surface expression of CD49d (avg 64% increase, p=0.03) and CXCR7 (avg 67% increase, p=0.02). Conversely, in vehicle control-treated 34-19+ LICs, stromal co-culture did not affect surface expression of the measured adhesion molecule receptors. In all 3 culture conditions, surface expression of CXCR4, CD49d, and CXCR7 was significantly higher in 34+38+19+ LICs compared to 34-19+ LICs (p≤0.01). We then measured apoptosis by flow cytometry and Annexin V binding. In the absence of chemotherapy, stromal co-culture significantly protected 34+38+19+ LICs from spontaneous apoptosis (p<0.001), but not 34-19+ LICs (p=0.16). Treatment with AMD3100 decreased stromal protection from spontaneous apoptosis in 34+38+19+ LICs (p<0.05), but not 34-19+ LICs (p=0.13). Notably, 34-19+ LICs were more resistant to spontaneous apoptosis even when cultured without stromal support (avg Annexin V-negative fraction 76.5% in 34-19+ vs. 62.8% in 34+38+19+, p=0.005). In spite of differences in stromal-mediated protection from spontaneous apoptosis, stromal co-culture protected both LIC populations from AraC- or Etop-induced apoptosis (p<0.001 for 34+38+19+, p<0.01 for 34-19+). Also, 34-19+ LICs were more resistant to Etop than 34+38+19+ LICs in the presence (p<0.001) or absence (p<0.01) of stroma. Finally, AMD3100 decreased stromal protection from AraC- or Etop-induced apoptosis (p<0.001 for 34+38+19+, p<0.05 for 34-19+). Conclusions: Our results suggest that the two immunophenotypically distinct LIC populations in infant MLL-R ALL may rely differently on the BM microenvironment. Higher surface expression of adhesion molecule receptors may explain why 34+38+19+ LICs derive more benefit from stromal co-culture, while lower surface expression of adhesion molecule receptors may explain decreased reliance on stroma by 34-19+ LICs. There may also be differences in chemotherapy resistance between the LIC populations. Despite these differences, CXCR4 inhibition enhanced chemosensitivity in both LIC populations. These results support the development of new therapeutic strategies that target leukemia-stroma interactions to improve outcomes for infants with MLL-R ALL. Disclosures No relevant conflicts of interest to declare.

Activity of Targeted Molecular Therapeutics Against Primary AML Cells: Putative Role of the Bone Marrow Microenvironment.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2304-2304 ◽  
Author(s):  
Teresa McQueen ◽  
Marina Konopleva ◽  
Michael Andreeff
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Culture System ◽  
Hematological Malignancies ◽  
Annexin V ◽  
Leukemic Cells ◽  
Mek Inhibitors ◽  
Molecular Therapeutics ◽  
Induced Apoptosis

Abstract In hematological malignancies, there are reciprocal interactions between leukemic cells and cells of the bone marrow (BM) microenvironment such as mesenchymal stem cells (MSC). It is speculated that specific BM niches may provide a sanctuary for subpopulations of leukemic cells to evade chemotherapy-induced death and allow acquisition of a drug-resistant phenotype. In this study, we compared anti-leukemia effects of Ara-C and various signal transduction and apoptosis inhibitors in a co-culture system of primary AML and human bone marrow-derived MSC. AML blasts from 11 primary AML samples with high (&gt;70%) blast count were co-cultured with MSC for 24 hours, after which they were exposed to the indicated concentrations of inhibitors for 48–96 hrs. Concentrations were selected on the basis of preliminary cell line studies which determined efficient inhibition of drug targets. Induction of apoptosis was analyzed by Annexin V flow cytometry after gating on the CD90 APC(−) (non-MSC) population. MSC protected leukemic blasts from spontaneous apoptosis in all 11 samples studied (mean annexinV positivity, 49.5±7.2% vs 25.3±4.8%, p&lt;0.001) and from Ara-C-induced cytotoxicity in 6 out of 11 samples (p=0.02). No difference in the degree of protection was noted when MSC from older vs. younger donors were used (not shown). Co-culture of leukemic cells with MSC resulted in significant (p&lt;0.03) suppression of inhibitor-induced apoptosis for all agents tested (Table 1), however PI3K/AKT inhibitors seemed to overcome MSC-mediated resistance. In addition, specific inhibitors of Bcl-2 and MDM2 induced apoptosis not only in suspension, but also in the MSC co-culture system, while Raf-1/MEK inhibitors were less effective. The AKT inhibitor A443654 caused apoptosis induction not only in leukemic cells, but also in MSC, which likely accounted for its high efficacy in stromal co-cultures (53±6% annexin V+). In a different study (Tabe et al, ASH 2005), we report that interactions of leukemic and BM stromal cells result in the activation of PI3K/ILK/AKT signaling in both, leukemic and stromal cells. We therefore propose that disruption of these interactions by specific PI3K/AKT inhibitors represents a novel therapeutic approach to eradicate leukemia in the BM microenvironment via direct effects on leukemic cells and by targeting activated BM stromal cells. Furthermore, Bcl-2 and MDM2 inhibitors appear to retain their efficacy in stroma-cocultured AML cells, while the efficacy of chemotherapy and Raf/MEK inhibitors in these conditions may be reduced. Further studies are aimed at the elucidation of the role of the BM microenvironment and its ability to activate specific signaling pathways in the pathogenesis of leukemias and on efforts to disrupt the MSC/leukemia interaction (Zeng et al, ASH 2005). Focus on this stroma-leukemia-stroma crosstalk may result in the development of strategies that enhance the efficacy of therapies in hematological malignancies and prevent the acquisition of a chemoresistant phenotype. Table 1. Leukemia Cell Apoptosis in a MSC/AML Co-Culture System Target Bcl-2/XL MDM2 PI3K AKT Raf-1 MEK Apoptosis was determined as percentage of Annexin V(+)CD90(−) cells, and calculated by the formula: % specific apoptosis = (test − control) x 100 / (100 − control). Compound, concentration Ara-C, 1 μM ABT-737, 0.1 μM Nutlin-3A, 2.5 μM LY294002, 10 μM A443654, 1 μM BAY43-9006, 2.5 μM CI1040, 3 μM AML 28±7 69±7 45±7 53.8±13.3 75±7 35±11 27±11 AML + MSC 16±4 38±6 28±6 31.2±6.9 53±6 18±8 15±5

Novel Role of HDAC Inhibitors in Bone Marrow Microenvironment: Activation of Leukemia Cell Phagocytosis through Annexin A1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2219-2219
Author(s):  
Yoko Tabe ◽  
Rooha Contractor ◽  
Susanne Radke ◽  
Michael Andreeff ◽  
Marina Konopleva
Keyword(s):  
Bone Marrow ◽  
Membrane Binding ◽  
Hdac Inhibitors ◽  
Leukemia Cell ◽  
Cdna Array ◽  
Bone Marrow Microenvironment ◽  
Leukemic Cells ◽  
Annexin A1 ◽  
Induced Apoptosis

Abstract Annexin A1 (ANX-A1) is a calcium-dependent membrane-binding protein involved in the modulation of apoptosis and phagocytosis (FASEB J.2003;17:1544). We have previously reported that HDAC inhibitor depsipeptide (FK228) caused marked growth inhibition and apoptosis in t(8;21) Kasumi-1 AML cells with up-regulation of 123 genes (by cDNA array) including ANX-A1 (3.5 fold; Tabe, Blood 2004). By chromatin immunoprecipitation (ChIP) assay, FK228 induced H4 and H3-K9 acetylation in the ANX-A1 promoter with corresponding induction of ANX-A1 mRNA (7.2±1.7 fold, TaqMan RT-PCR) and protein (western blot analysis). The markedly increased ANX-A1 protein localized on the cell membrane of Kasumi-1 cells exposed to FK228 was confirmed by immunofluorecence analysis using confocal microscopy. ANX-A1 membrane localization was diminished by treatment with anti-ANX-A1 mAb. To investigate the contribution of ANX-A1 to FK228-induced apoptosis, we neutralized ANX-A1 by anti-ANX-A1 mAb. This moderately decreased FK228 induced apoptosis (36.0±4.1 vs 26.5±3.7% AnnexinV(+)/PI(+) cells, p=0.01). Similarly, Kasumi-1 cells transfected with siRNA/ANX-A1 were less sensitive to FK228-induced cell death compared with nonsense (N) siRNA transfected cells (siRNA 31.2±3.1% vs NsiRNA 39.5±2.9% annexin(+) cells, p=0.03). These data indicate that the upregulation of endogeneous ANX-A1 (either membrane-binding or secreted form) promotes cell apoptosis in an autocrine fashion. Next, we investigated the functional role of ANX-A1 on leukemia cell phagocytosis. The engulfment of Kasumi-1 cells by cocultured human THP-1 monocyte-derived macrophages was evaluated by cell adherence assay. Compared with untreated cells, the exposure to FK228 induced a dramatic increase in Kasumi-1 cells attachment to macrophages (untreated vs FK228 treated; 57 ± 9 cells vs 196 ± 33 cells/ microscopic fields (0.08 mm2/field), n = 5; p=0.01). FK228-induced cell attachment was completely abrogated in the siRNA/ANX-A1 transfected Kasumi-1 cells (60.5% ± 10.5% decrease; n = 5; p<0.001). Consistently, co-treatment with FK228 and anti-ANX-A1 mAb followed by washout of both compounds resulted in significantl repression of FK228-stimulated engulfment of leukemic cells by macrophages (54.1% ± 3.0% decrease; n = 5; p=0.02). This effect was not further enhanced by adding anti-ANX-A1 mAb to the co-culture medium, suggesting that membrane-associated but not soluble ANX-A1 contributes to leukemia cell engulfment by macrophages. Results presented here demonstrate a novel mechanism of action of HDAC inhibitors in the context of bone marrow microenvironment via histone acetylation, increased expression and externalization of ANX-A1, which provides an “eat-me” signal and mediates phagocytic clearance of apoptotic leukemic cells by macrophages. Our data further suggest that ANX-A1 is silenced via histone deacetylation in leukemic cells, and its re-expression by HDAC inhibitors may stimulate apoptosis in an autocrine fashion while diminishing the inflammatory response through activating phagocytosis in the bone marrow microenvironment.

Protection of ALL Cells by Bone Marrow Stromal Cells and the Underlying Molecular Mechanism.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2587-2587
Author(s):  
Yang Yang ◽  
Baohua Sun ◽  
Saradhi Mallampati ◽  
Zhen Cai ◽  
Xiaoping Sun
Keyword(s):  
Bone Marrow ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Stromal Cells ◽  
Cell Apoptosis ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Leukemic Cells ◽  
New Therapies ◽  
Induced Apoptosis

Abstract Abstract 2587 Acute lymphoblastic leukemia (ALL) is one of the fastest-growing hematological malignancies affecting patients with all ages, particularly children. Significant advances have been made in recent years in our understanding of the disease and the development of new therapies, which have led to a greatly improved outcome. Nevertheless, in a significant number of patients with ALL, the disease relapse and become resistant to treatment, causing death of the patients. Increasing evidence suggests that relapse of the disease and resistant to treatment are largely attributed to the protection of the leukemic cells by various components in the microenvironment, such as bone marrow stromal cells. However, the cross-talk between leukemic cells and their microenvironment remains poorly understood. Therefore, better understanding the mechanisms underlying the protection of ALL cells by the microenvironment is of ultimate importance in developing new therapies targeting such protection and eventually eradicating all the leukemic cells to cure the disease. In this study, we used a coculture system with leukemic cells and bone marrow stromal cells (MSC) to mimic the in vivo interaction between the two cell types to explore the molecular events that might be responsible for the protection of ALL cells from Ara-C induced apoptosis. We cocultured human primary ALL cells with hTERT-immortalized normal human MSC and evaluated ALL cell apoptosis by FACS after staining with Annexin V and propidium iodide. In all 8 cases, the MSC provided significant protection of ALL cells from both spontaneous and Ara-C induced apoptosis. For example, the mean Ara-C induced apoptosis of ALL cells cultured without MCS was 42.7% (range, 27–54%), whereas it was 19.1% (range, 8–27%) with MSC. Similar results were obtained with human leukemia cell lines Reh, SEMK2 and RS4.11. We also found that the murine MSC line M210B4 could provide similar protection to ALL cells, whether the ALL cells are primary or cell lines. The reduced apoptosis in the coculture were confirmed by Western blot which showed that MSC could protect ALL cells from Caspase-3 and PARP cleavage. Furthermore, our results showed no significant Ara-C induced reduction in S phase when cocultured with MSC. This phenomenon was associated with decreased cyclinA and CDK2 expression. In addition, we found that cocultured with MSC resulted in phosphorylation of AKT in ALL cells and PI3K inhibitor LY294002 specifically inhibited MSC-induced activation of AKT and promoted ALL cell apoptosis. In addition, beta-catenin and c-myc had increased expression in ALL cells cocultured with MSC, suggesting that Wnt pathway could play a role in MSC-mediated protection. To identify candidate molecules potentially involved in the protection of ALL cells by MSC, we performed gene expression microarray analyses with ALL cells exposed to Ara-C in presence or absence of MSC. Our data indicated that several signaling pathways might be involved in this process, including apoptosis signaling and cell cycle checkpoint control, which confirmed above findings. The top expressed genes identified in the microarray studies were confirmed by RT-PCR. Collectively, our results demonstrated that MSC can protect ALL cells from Ara-C induced apoptosis by multiple signaling pathways, such as those involving PI3K/AKT and Wnt signaling. Hence, targeting these pathways may become potential novel therapeutic strategies to disrupt the support of the microenvironment to ALL cells and to eventually eradicate leukemic cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Stromal CYR61 Confers Resistance to Mitoxantrone Via Spleen Tyrosine Kinase Activation in Human Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2228-2228
Author(s):  
Xin Long ◽  
Laszlo Perlaky ◽  
Tsz-Kwong Chris Man ◽  
Michele S. Redell
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Stromal Cell ◽  
Chemotherapy Resistance ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a life-threatening bone marrow malignancy with a relapse rate near 50% in children, despite aggressive chemotherapy. Accumulating evidence shows that the bone marrow stromal environment protects a subset of leukemia cells and allows them to survive chemotherapy, eventually leading to recurrence. The factors that contribute to stroma-induced chemotherapy resistance are largely undetermined in AML. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in human AML cells. We used two human bone marrow stromal cell lines, HS-5 and HS-27A, to study stroma-induced chemotherapy resistance. Both stromal cell lines are equally effective in protecting AML cell lines and primary samples from apoptosis induced by chemotherapy agents, including mitoxantrone, etoposide, and cytarabine. By gene expression profiling using the Affymetrix U133Plus 2 platform, we previously found that CYR61 was among the genes that were commonly upregulated in AML cells by both stromal cell lines. CYR61 is a secreted matricellular protein that is expressed at relatively low levels by AML cells, and at higher levels by stromal cells. CYR61 binds and activates integrins and enhances growth factor signaling in AML cells, and it has been associated with chemoresistance in other malignancies. Our current data provide functional evidence for a role for this protein in stroma-mediated chemoresistance in AML. First, we added anti-CYR61 neutralizing immunoglobulin (Ig), or control IgG, to AML-stromal co-cultures, treated with chemotherapy for 24 hours, and measured apoptosis with Annexin V staining and flow cytometry. In THP-1+HS-27A co-cultures treated with 50 nM mitoxantrone, the apoptosis rate was 33.0 ± 3.7% with anti-CYR61 Ig v. 16.3 ± 4.2% with control IgG; p=0.0015). Next, we knocked down CYR61 in the HS-5 and HS-27A stromal cell lines by lentiviral transduction of two individual shRNA constructs, and confirmed knockdown (KD) at the gene and protein levels for both cell lines. These CYR61-KD stromal cells provided significantly less protection for co-cultured AML cells treated with mitoxantrone, compared to stromal cells transduced with the non-silencing control. For example, the apoptosis rate for THP-1 cells co-cultured with CYR61-KD HS-27A cells was 10.8 ± 0.8%, compared to 6.8 ± 1.1% for THP-1 cells co-cultured with control HS-27A cells (p=0.02). Similar results were obtained with NB-4 AML cells. These results demonstrate that CYR61 contributes to stroma-mediated chemoresistance. CYR61 binds to integrin αvβ3 (Kireeva, et al, J. Biol. Chem., 1998, 273:3090), and this integrin activates spleen tyrosine kinase (Syk) (Miller, et al, Cancer Cell, 2013, 24:45). Using intracellular flow cytometry, we found that activated Syk (pSyk) increased in THP-1 and NB-4 cell lines, and in primary AML patient samples, upon exposure to control HS-27A cells. In primary samples, the mean fluorescence intensity (MFI) for pSyk averaged 11.7 ± 1.3 in co-culture v. 6.6 ± 0.6 for cells cultured alone (p=0.004, n=10). In contrast, pSyk did not significantly increase in AML cells co-cultured with CYR61-KD HS-27A cells (MFI for primary patient samples: 8.6 ± 0.8). This result implicates Syk as a downstream signaling mediator of CYR61. To determine the role of CYR61-induced Syk signaling in chemotherapy resistance, we treated AML-stromal cell co-cultures with 3 uM R406, a potent Syk inhibitor, or DMSO, then added 300 nM mitoxantrone, and measured apoptosis after 24 hours. In AML cells co-cultured with control HS-27A cells, mitoxantrone-induced apoptosis was significantly increased by Syk inhibition (THP-1 cells: 13.7 ± 0.7% with R406 v. 10.0 ± 0.3% with DMSO, p<0.05), consistent with reduced chemoresistance. Notably, R406 did not further increase mitoxantrone-induced apoptosis in AML cells co-cultured with CYR61-KD HS-27A stromal cells (THP-1 cells: 15.7 ± 0.2% with R406 v. 16.9 ± 0.4% with DMSO). Similar results were seen with NB-4 cells, as well. These results support the notion that CYR61 signals through the integrin-Syk pathway to protect AML cells from chemotherapy. Therefore, the CYR61 - integrin - Syk pathway may be a potential therapeutic target for overcoming stroma-induced chemotherapy resistance in AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inhibition of BMP-Smad Pathway Reduces Leukemic Stemness in Pediatric AML

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3731-3731
Author(s):  
Xin Long ◽  
Chris Man ◽  
Padmini Narayanan ◽  
Elizabeth Seashore ◽  
Michele Redell
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cells ◽  
Chemotherapy Resistance ◽  
Reverse Direction ◽  
Self Renewal ◽  
Smad Pathway ◽  
Viable Cells ◽  
Pediatric Aml ◽  
Induced Apoptosis

Despite aggressive chemotherapy, relapse occurs in almost half of children with acute myeloid leukemia (AML), with very dismal survival. Novel and mechanism-driven therapies are desperately needed to conquer chemotherapy resistance and leukemic stemness in pediatric AML. Within the bone marrow niche, stromal cells protect leukemia cells from chemotherapy, maintain leukemic stemness, and eventually lead to disease recurrence. We developed an in vitro AML cell-stromal cell co-culture model to mimic bone marrow microenvironment. Stroma-leukemia cell interaction leads to activation of various signaling molecules in AML cells that allow them to evade apoptosis. One such example is extracellular signal-regulated kinases 1/2 (ERK1/2), important pro-survival proteins. ERK1/2 are activated by the Ras/Raf/ mitogen-activated protein kinase kinase (MAPK/ERK kinase or MEK) pathway downstream of signals from the stroma. We recently showed that stromal co-culture activates ERK1/2 in pediatric AML samples, contributing to chemotherapy resistance (Long, et al, 2017, Oncotarget, 8:90037). To identify genes that are regulated in AML cells by ERK1/2 activation, 4 pediatric AML samples were cultured alone, or co-cultured with mOrange-expressing stroma for 24 hours, in the presence or absence of a selective MEK inhibitor, selumetinib (1 μM). Thereafter, cells were flow sorted to exclude mOrange+ stroma and CD45high/SSClow lymphocytes. Sorted AML cells underwent total RNA extraction for nCounter® PanCancer Pathways Panel (Nanostring Technologies) gene expression profiling study. We focused on the genes that were either up- or down-regulated by co-culture with stroma, and changed in the reverse direction by the addition of selumetinib. We chose a few genes among the list (BMP2, BNIP3, H2AFX, DUSP2, FZD3, BCL2L1, CHEK2) that are reported to be involved in oncogenesis, chemotherapy resistance, cell growth and survival. Using qRT-PCR, we confirmed bone morphogenic protein 2 (BMP2) to be upregulated in AML cells by stroma, and the effect of stroma was reversed by selumetinib. Further, we confirmed the same change of BMP2 at the protein level by FACS. Smad1, 5 and 8 are transcriptional factors immediately downstream from BMP receptors and play a central role in BMP signal transduction. Using FACS we discovered stroma-induced activation of Smad 1/8 in pediatric AML patient samples, which was partially alleviated by selumetinib and a selective BMP inhibitor, K02288 (10 μM). BMPs are growth factors that belong to the transforming growth factor beta (TGF-beta) superfamily and are thought to be involved in stem cell properties such as self-renewal. To determine if the BMP-Smad pathway plays a role in chemotherapy resistance, 4 pediatric AML patient samples were cultured on or off stromal cells, and treated with cytarabine (10 μM) with or without K02288 (10 μM) for 24h. Cells were analyzed for cytarabine-induced apoptosis with Annexin V staining by FACS, excluding stromal cells and lymphocytes. K02288 treatment did not alter cytarabine-induced apoptosis. We next tested the potential role of BMP-Smad pathway in leukemic self-renewal in pediatric AML samples. Pediatric AML patient samples were plated at 50,000/ml or 100,000/ml in Methocult medium (H-4535) to quantify stem and progenitor cells. Samples were treated with vehicle or K02288 (10 μM). Colonies and viable cells were counted and normalized to control 7-14 days after plating. Harvested cells were stained for CD34, CD14 and CD11b to evaluate differentiation by FACS. Remaining cells were replated at the same density for 2 more rounds. We found that K02288 reduced colony counts (e.g., 100±0% in vehicle control, v. 25±10% in K02288, n=5, p<0.002, post 2nd plating) and decreased the number of viable cells at 2nd and 3rd plating (Figure 1). The BMP inhibitor also promoted differentiation of leukemic stem cell in pediatric AML samples, as evidenced by increased CD14 and CD11b expression. In contrast, for normal pediatric bone marrow samples, K02288 did not change colony counts or the number of viable cells, and it did not promote differentiation. Those data suggested BMP-Smad is likely to contribute to leukemic stemness in pediatric AML without disrupting normal hematopoietic stem cells. Therefore, BMP-Smad pathway may be a promising therapeutic target to reduce leukemia burden and improve survival for children with AML. Disclosures No relevant conflicts of interest to declare.

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