scholarly journals CXCR4 Inhibition Enhances Efficacy of FLT3 Inhibitors in FLT3-Mutated AML Augmented by Suppressed TGF-β Signaling

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1737
Author(s):  
Bo-Reum Kim ◽  
Seung-Hyun Jung ◽  
A-Reum Han ◽  
Gyeongsin Park ◽  
Hee-Je Kim ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Kinase Inhibitor ◽  
Mapk Pathway ◽  
Combined Treatment ◽  
Leukemic Cells ◽  
Transient Effects ◽  
Flt3 Inhibitors ◽  
Induced Apoptosis ◽  
Cxcr4 Axis

Given the proven importance of the CXCL12/CXCR4 axis in the stroma–acute myeloid leukemia (AML) interactions and the rapid emergence of resistance to FLT3 inhibitors, we investigated the efficacy and safety of a novel CXCR4 inhibitor, LY2510924, in combination with FLT3 inhibitors in preclinical models of AML with FLT3-ITD mutations (FLT3-ITD-AML). Quizartinib, a potent FLT3 inhibitor, induced apoptosis in FLT3-ITD-AML, while LY2510924 blocked surface CXCR4 without inducing apoptosis. LY2510924 significantly reversed stroma-mediated resistance against quizartinib mainly through the MAPK pathway. In mice with established FLT3-ITD-AML, LY2510924 induced durable mobilization and differentiation of leukemia cells, resulting in enhanced anti-leukemia effects when combined with quizartinib, whereas transient effects were seen on non-leukemic blood cells in immune-competent mice. Sequencing of the transcriptome of the leukemic cells surviving in vivo treatment with quizartinib and LY2510924 revealed that genes related to TGF-β signaling may confer resistance against the drug combination. In co-culture experiments of FLT3-ITD-AML and stromal cells, both silencing of TGF-β in stromal cells or TGF-β-receptor kinase inhibitor enhanced apoptosis by combined treatment. Disruption of the CXCL12/CXCR4 axis in FLT3-ITD-AML by LY2510924 and its negligible effects on normal immunocytes could safely enhance the potency of quizartinib, which may be further improved by blockade of TGF-β signaling.

scholarly journals Targeting of mTORC1/2 by the mTOR kinase inhibitor PP242 induces apoptosis in AML cells under conditions mimicking the bone marrow microenvironment

Blood ◽  
2012 ◽  
Vol 120 (13) ◽  
pp. 2679-2689 ◽  
Author(s):  
Zhihong Zeng ◽  
Yue Xi Shi ◽  
Twee Tsao ◽  
YiHua Qiu ◽  
Steven M. Kornblau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Kinase Inhibitor ◽  
Mammalian Target Of Rapamycin ◽  
Leukemic Cells ◽  
Phosphatidylinositol 3 Kinase ◽  
Mtor Kinase ◽  
Phosphorylated Akt ◽  
Induced Apoptosis

Abstract The interactions between the bone marrow (BM) microenvironment and acute myeloid leukemia (AML) is known to promote survival of AML cells. In this study, we used reverse phase-protein array (RPPA) technology to measure changes in multiple proteins induced by stroma in leukemic cells. We then investigated the potential of an mTOR kinase inhibitor, PP242, to disrupt leukemia/stroma interactions, and examined the effects of PP242 in vivo using a mouse model. Using RPPA, we confirmed that multiple survival signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), were up-regulated in primary AML cells cocultured with stroma. PP242 effectively induced apoptosis in primary samples cultured with or without stroma. Mechanistically, PP242 attenuated the activities of mTORC1 and mTORC2, sequentially inhibited phosphorylated AKT, S6K, and 4EBP1, and concurrently suppressed chemokine receptor CXCR4 expression in primary leukemic cells and in stromal cells cultured alone or cocultured with leukemic cells. In the in vivo leukemia mouse model, PP242 inhibited mTOR signaling in leukemic cells and demonstrated a greater antileukemia effect than rapamycin. Our findings indicate that disrupting mTOR/AKT signaling with a selective mTOR kinase inhibitor can effectively target leukemic cells within the BM microenvironment.

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.

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.

SDF-1 Inhibition Using Spiegelmer® Nox-A12 As a Novel Strategy For Targeting AML Cells Within Their BM Microenvironment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2454-2454 ◽  
Author(s):  
Rodrigo Jacamo ◽  
Zhihong Zeng ◽  
Ye Chen ◽  
Yuexi Shi ◽  
Teresa McQueen ◽  
...  
Keyword(s):  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Cell Homing ◽  
Bm Niche ◽  
Survival Signaling ◽  
Cell Mobilization ◽  
Induced Apoptosis ◽  
Cxcr4 Axis

Abstract Bone marrow (BM) mesenchymal stromal cells (MSC) protect leukemia cells from chemotherapy-induced apoptosis. CXCR4, the receptor for the stromal-derived factor 1 (SDF-1/CXCL12), is involved in modulating leukemic progenitor cell homing and it is essential for the migration of these cells to the BM niche. As the BM niche imparts favorable survival advantage to leukemia cells, a strategy to disrupt leukemic cell homing to the BM microenvironment may represent a novel way to effectively kill these malignant cells. Strategies to disrupt the SDF-1/CXCR4 axis have relied on inhibition of the CXCR4 receptor using various drugs including plerixafor, which is potent at mobilizing normal progenitor cells. We previously found that plerixafor antagonized SDF-1 and MSC-induced migration of leukemic cells, inhibited SDF-1-induced AKT and ERK activation, and decreased stroma-mediated protection from AraC-induced apoptosis in 11 out of 18 primary AML samples (Zeng et al Blood 2009; 113: 6215). We also found that the inhibition of CXCR4 induces miRNA let-7a expression via the transcription factor Yin Yang1 (Chen et al, JCI, 2013; 123:2395), and induces chemosensitization irrespective of cell mobilization. While inhibition of CXCR4 has shown promise for use in AML therapy (Andreeff, et al ASH, Blood 120(21) (#142), 11/2012), a more effective strategy might be to target the SDF-1 ligand itself since SDF-1 can bind to other receptors (i.e. CXCR7) that are likely not recognized by CXCR4 antibodies. NOXXON (Berlin, Germany) has recently identified a potent inhibitor to SDF-1 that is based on a so-called Spiegelmer®. The Spiegelmer NOX-A12 is a structured l-RNA oligonucleotide, PEGylated at the 5'-end to improve the pharmacokinetics. In healthy volunteers, NOX-A12 displays a half-life of∼38 hours and has demonstrated a long-term and dose-dependent mobilization of WBCs and CD34+ cells. The drug is currently tested in clinical trials in multiple myeloma and chronic lymphocytic leukemia patients (Gobbi et al., ASH 2012 120: 4593). In the current study, the ability of NOX-A12 to suppress survival signaling in leukemic and MSC cells was examined. In addition, we tested the ability of the Spiegelmer to mobilize leukemia cells in a murine xenograft model. NOX-A12 was found to suppress the phosphorylation of ERK in AML cells, and suppress the phosphorylation of ERK, FAK, and STAT3 in MSC. These results suggested that SDF-1 affected diverse survival signaling pathways in the leukemic cells and the critical supporting stromal niche. In vivo activity was examined in NOD/SCID/-g- mice injected intravenously with murine leukemia Baf/ITDluc/GFP cells and monitored by a non-invasive bioluminescent imaging. The in vivo mobilization assay was assessed when the majority of mice had approximately 10% tumor burden. Plerixafor, NOX-A12, or a combination of both agents was subcutaneously administrated and leukemic GFP+ cells were detected by analytical flow cytometry. Plerixafor and NOX-A12 administered separately increased total WBC count by 2- to 3-fold and concomitantly increased the percentage of circulating GFP+ cells in all mice (to 14.2% and 18.5%, respectively). Importantly, the combination of both targeted agents further increased the percentage of circulating GFP+ cells in an approximately additive fashion (to 26.4%). Conclusion we demonstrate that the novel SDF-1 Spiegelmer NOX-A12 can suppress survival signaling in both leukemia and supportive MSC cells. Furthermore, increased mobilization of leukemic cells in vivo was observed when NOX-A12 and plerixafor were used in combination. These findings suggest the possibility that targeted disruption of leukemia/stroma interactions by optimized blockade of the pro-survival SDF-1/CXCR4 axis will not only increase leukemia cell mobilization, but also enhance the sensitivity to chemotherapy of both mobilized and non-mobilized the leukemic cells and leukemic stem cells. Experiments are ongoing to further validate this hypothesis. Disclosures: Kruschinski: Noxxon: Employment.

Differentiation arrest and stromal cell-independent growth of murine erythroleukemia cells are associated with elevated expression of ets-related genes but not with mutation of p53

1993 ◽  
Vol 13 (9) ◽  
pp. 5582-5592
Author(s):  
R J Nibbs ◽  
K Itoh ◽  
W Ostertag ◽  
P R Harrison
Keyword(s):  
Stromal Cells ◽  
Stromal Cell ◽  
Gene Activation ◽  
Leukemic Cells ◽  
Term Survival ◽  
Elevated Expression ◽  
D Cells ◽  
Murine Erythroleukemia

The ELM erythroleukemia is novel in that long-term survival of leukemic cells in culture (ELM-D cells) is dependent on contact with a bone marrow-derived stromal feeder cell layer. However, a number of stroma-independent (ELM-I) mutants that vary in their ability to differentiate in vitro in response to erythropoietin and interleukin-3 have been derived. We have attempted to define the genetic changes responsible for these different phenotypes. At the p53 locus in the primary leukemic cells, one copy of the gene has been lost whereas the other contains an 18-bp depletion, implicating its mutation as an early step in the development of the leukemia. Changes in ets gene expression have also been found. The Fli-1 gene region is rearranged in the primary tumor because of the insertion of a retrovirus inserted upstream of one Fli-1 allele, but this does not result in Fli-1 gene activation in any of the ELM-D or ELM-I cell lines except one. It seems significant that this line is the only one to have lost the ability to differentiate in response to erythropoietin. In addition, up-regulation of erg is associated with stromal cell-independent growth, since all ELM-I mutants have moderate levels of erg mRNA, whereas only low or undetectable levels are found in primary leukemic cells in vivo or in ELM-D cells in vitro. This up-regulation of erg mRNA seems to be important for stromal cell-independent growth, since ELM-D cells show elevated expression of the erg gene after separation from stromal cells. This seems to be made permanent in ELM-I mutants, since they do not down-regulate erg mRNA when grown in contact with stromal cells. We therefore propose that ets family members regulate both the survival and differentiation of erythroid cells.

Rapamycin sensitizes multiple myeloma cells to apoptosis induced by dexamethasone

Blood ◽  
2004 ◽  
Vol 103 (8) ◽  
pp. 3138-3147 ◽  
Author(s):  
Thomas Strömberg ◽  
Anna Dimberg ◽  
Anna Hammarberg ◽  
Kristina Carlson ◽  
Anders Österborg ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Kinase Inhibitor ◽  
Mammalian Target Of Rapamycin ◽  
G1 Arrest ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Induced Apoptosis ◽  
Molecular Mode

Abstract Circumvention of chemoresistance in the B-cell neoplasm multiple myeloma (MM) might be achieved by targeting certain intracellular signaling pathways crucial for survival of the malignant clone. The use of the macrolide rapamycin, selectively inhibiting the phosphoprotein mammalian target of rapamycin (mTOR) downstream of, for example, insulin-like growth factor-I receptor (IGF-IR), possibly represents such a molecular mode of therapy. By using a panel of MM cell lines we showed that rapamycin induced G0/G1 arrest, an effect being associated with an increase of the cyclin-dependent kinase inhibitor p27 and a decrease of cyclins D2 and D3. Interestingly, in primary, mainly noncycling MM cells, rapamycin, at clinically achievable concentrations, induced apoptosis. More important, rapamycin sensitized both MM cell lines and primary MM cells to dexamethasone-induced apoptosis. This effect was associated with a decreased expression of cyclin D2 and survivin. The phosphorylation of the serine/threonine kinase p70S6K at Thr389 and Thr421/Ser424 was down-regulated by rapamycin and/or dexamethasone. Strikingly, the combinatorial treatment with rapamycin and dexamethasone suppressed the antiapoptotic effects of exogenously added IGF-I and interleukin 6 (IL-6) as well as their stimulation of p70S6K phosphorylation. The induction of apoptosis by rapamycin and dexamethasone despite the presence of survival factors was also demonstrated in primary MM cells, thus suggesting this drug combination to be active also in vivo. (Blood. 2004;103:3138-3147)

Phosphorylation of Argonaute 2 at serine-387 facilitates its localization to processing bodies

2008 ◽  
Vol 413 (3) ◽  
pp. 429-436 ◽  
Author(s):  
Yan Zeng ◽  
Heidi Sankala ◽  
Xiaoxiao Zhang ◽  
Paul R. Graves
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Kinase Inhibitor ◽  
Mapk Pathway ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Processing Bodies ◽  
Mitogen Activated Protein

Ago (Argonaute) proteins are essential effectors of RNA-mediated gene silencing. To explore potential regulatory mechanisms for Ago proteins, we examined the phosphorylation of human Ago2. We identified serine-387 as the major Ago2 phosphorylation site in vivo. Phosphorylation of Ago2 at serine-387 was significantly induced by treatment with sodium arsenite or anisomycin, and arsenite-induced phosphorylation was inhibited by a p38 MAPK (mitogen-activated protein kinase) inhibitor, but not by inhibitors of JNK (c-Jun N-terminal kinase) or MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]. MAPKAPK2 (MAPK-activated protein kinase-2) phosphorylated bacterially expressed full-length human Ago2 at serine-387 in vitro, but not the S387A mutant. Finally, mutation of serine-387 to an alanine residue or treatment of cells with a p38 MAPK inhibitor reduced the localization of Ago2 to processing bodies. These results suggest a potential regulatory mechanism for RNA silencing acting through Ago2 serine-387 phosphorylation mediated by the p38 MAPK pathway.

Constitutive FLT3 Activation Results in Specific Changes in Gene Expression in Myeloid Leukemic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1115-1115
Author(s):  
Kyu-Tae Kim ◽  
Kristin Baird ◽  
Sean Davis ◽  
Mark Levis ◽  
Obdulio Piloto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Microarray Analysis ◽  
Cdna Microarray ◽  
Mapk Pathway ◽  
Cyclin D3 ◽  
Leukemic Cells ◽  
Cdna Microarray Analysis ◽  
Flt3 Inhibitors ◽  
Before And After

Abstract Constitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 play an important role in leukemogenesis. They are the most common genetic alteration in AML and their presence is associated with poor prognosis. To better understand FLT3 signaling in leukemogenesis, we have examined the changes in gene expression induced by FLT3/ITD or constitutively activated wild type FLT3 signaling by cDNA microarray analysis. In order to minimize gene expression changes that might be drug specific and not related to FLT3 inhibition or might be cell-type specific, we used three different FLT3 inhibitors, CEP-701, CEP-5214, and AG1296, and three different constitutively activated FLT3-expressing leukemia-derived cell lines, EOL-1, MOLM-14 and MV4-11. We considered to be FLT3 responsive only those genes whose expression consistently changed in response to FLT3 inhibition by each of the three FLT3 inhibitors in all of the cell lines. RNA for hybridization to the microarrays was harvested from cells both before and after increasing times of FLT3 inhibition to determine genes which decreased or increased in response to FLT3 signaling. In addition, because the inhibitors are all reversible, RNA was also harvested from the cells at increasing times after release from FLT3 inhibition. This enabled us to confirm that, for example, genes whose expression appeared FLT3 dependent and were thus down-regulated by FLT3 inhibition, returned towards normal levels after FLT3 signaling was allowed to resume. Statistical analysis of the microarray results indicated a limited set of genes are highly and consistently affected by FLT3 inhibition and return toward pretreatment levels after release from inhibitor. We confirmed the cDNA microarray data using quantitative real-time PCR. Several of the most significantly affected genes are involved in the Ras/MAPK pathway including DUSP6, DUSP7, MAPK6, TNF, and cMyc. Other sets of genes are involved in JAK/STAT or Wnt signaling pathways including Pim-1, cMyc, Cyclin D3, IL4 receptor, and CISH. These genes are all consistently down-regulated after FLT3 inhibition. These data further confirm the role of constitutively activating FLT3 in mediating multiple signal transduction pathways. We also found several transcriptional factors (RUNX1/AML1, MAFG, XBP1, TGFBI4, and BRD8), several genes involved in receptor-mediated signaling (IL1RAP, CDC42EP3, PLAUR, LY64), and several genes involved in cell proliferation, metabolism, or structure (BCL7A, APTX, GHRH, SET7, ATAD2, CLIC1, CRIP2, MRPL12, VIM) to be consistently differentially expressed. In summary, we have found by cDNA microarray analysis and confirmed by QPCR, a consistent pattern of FLT3 dependent gene expression. The alteration of the gene expression profile in these cells is likely the mechanism of FLT3-mediated leukemogenisis.

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 (>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<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<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

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.

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