Extended Exposure To The CXCR4 Inhibitor Plerixafor May Lead To Enhanced Microenvironment Interactions in Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1295-1295
Author(s):  
Edward Allan R. Sison ◽  
Daniel Magoon ◽  
Patrick Brown
Keyword(s):  
Bone Marrow ◽  
Adhesion Molecules ◽  
Cell Lines ◽  
Prolonged Exposure ◽  
Surface Expression ◽  
Cxcr4 Expression ◽  
Antibody Binding ◽  
Drug Binding ◽  
Dose Dependent ◽  
Extended Exposure

Abstract Background We have previously demonstrated that inhibition of CXCR4 in ALL decreases CXCR4 antibody binding, inhibits SDF-1α-(CXCL12)-induced chemotaxis, and overcomes chemotherapy resistance conferred by the bone marrow microenvironment. Specifically, we found that treatment with plerixafor and araC significantly decreased leukemic burden in a xenograft model of infant ALL, compared to treatment with araC alone. In those experiments, we treated mice on 3 consecutive days per week for 2 weeks with plerixafor and araC. However, the combination did not eradicate the leukemia in our model. We hypothesized that extended exposure to plerixafor may have led to increased interactions between surviving leukemic blasts and the bone marrow microenvironment. In our current experiments, we sought to characterize the effects of prolonged exposure to plerixafor in ALL. Methods/Results We treated pre-B (HB-1119, Nalm-6) and T (CCRF-CEM-1301, Jurkat) ALL cell lines with a dose range of plerixafor and harvested cells for FACS over an extended time course. We measured surface CXCR4 (s-CXCR4) expression using 3 antibodies: 12G5, which attaches to the SDF-1α and drug-binding site of CXCR4, and 1D9 and 2B11, which do not compete with SDF-1α or drug binding. 12G5 binding was decreased by plerixafor even at 1 hour and this effect was concentration-dependent. Interestingly, we found a time and dose-dependent increase in 1D9 and 2B11 antibody binding, suggesting that plerixafor caused an actual increase in s-CXCR4 over time. Increases in 1D9 and 2B11 binding were inversely proportional to decreases in 12G5 binding. We also measured surface expression of CD49d (VLA-4), which binds to fibronectin and VCAM-1; CXCR7, which binds to SDF-1α and CXCL11; and CXCR3, which binds to CXCL9, 10, and 11. We hypothesized that CXCR4 inhibition would lead to upregulation of parallel pathways of leukemia-stroma interactions. CD49d was highly expressed at baseline, while CXCR7 and CXCR3 were expressed to a lesser degree. Treatment with plerixafor led to dose-dependent increases in CXCR7 and variable changes in CD49d and CXCR3 surface expression, suggesting that plerixafor can modulate surface expression of adhesion molecules other than CXCR4. Next, we treated ALL cell lines with plerixafor (0, 10, 100 nM) for 72 hours, washed with PBS, and resuspended the cells in fresh medium to determine the effects of extended exposure to plerixafor and subsequent withdrawal. First, we measured surface expression of s-CXCR4 after 72 hours of treatment with plerixafor and found that 12G5 binding was decreased, while 1D9/2B11 binding was increased in an inversely proportional manner. After withdrawal, 12G5 binding increased to untreated levels between 4 and 24 hours, while 1D9/2B11 binding decreased to untreated levels between 4 and 72 hours. We also measured surface expression of CD49d, CXCR7, and CXCR3 and found that the effects of plerixafor treatment and withdrawal were variable by cell line. For example, after plerixafor treatment, surface expression of CD49d and CXCR7 was increased in Nalm-6 and surface expression of CXCR7 and CXCR3 was increased in CCRF-CEM-1301. Interestingly, 4 hours after plerixafor withdrawal, CD49d expression was increased in Jurkat and Nalm-6, and CXCR7 expression was increased in CCRF-CEM-1301, HB-1119, and Jurkat. Finally, we measured migration of washed cells from each treatment condition through a permeable membrane toward medium containing SDF-1α or medium alone. Despite CXCR4 inhibition for 72 hours, all plerixafor-treated cells migrated in response to SDF-1α. In addition, some plerixafor-treated cells exhibited significantly increased SDF-1α-induced chemotaxis compared to control-treated cells. These findings imply that increases in s-CXCR4 induced by 72 hours of treatment with plerixafor are functional. Conclusions Treatment of ALL cell lines with plerixafor led to a dose-dependent decrease in 12G5 antibody binding with a simultaneous overall increase in s-CXCR4 expression. Prolonged exposure to plerixafor led to increased s-CXCR4 expression that persisted for up to 72 hours after drug withdrawal, modulated surface expression of additional adhesion molecules, and enhanced SDF-1α-induced chemotaxis. Therefore, additional careful studies of CXCR4 inhibitors and other microenvironment-targeted agents must be performed in order to determine their optimal use in ALL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Photon versus carbon ion irradiation: immunomodulatory effects exerted on murine tumor cell lines

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Laura Hartmann ◽  
Philipp Schröter ◽  
Wolfram Osen ◽  
Daniel Baumann ◽  
Rienk Offringa ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Ion Irradiation ◽  
Dose Range ◽  
Cancer Cell Line ◽  
Surface Expression ◽  
Photon Radiation ◽  
Immunomodulatory Effects ◽  
Carbon Ion ◽  
Dose Dependent

AbstractWhile for photon radiation hypofractionation has been reported to induce enhanced immunomodulatory effects, little is known about the immunomodulatory potential of carbon ion radiotherapy (CIRT). We thus compared the radio-immunogenic effects of photon and carbon ion irradiation on two murine cancer cell lines of different tumor entities. We first calculated the biological equivalent doses of carbon ions corresponding to photon doses of 1, 3, 5, and 10 Gy of the murine breast cancer cell line EO771 and the OVA-expressing pancreatic cancer cell line PDA30364/OVA by clonogenic survival assays. We compared the potential of photon and carbon ion radiation to induce cell cycle arrest, altered surface expression of immunomodulatory molecules and changes in the susceptibility of cancer cells to cytotoxic T cell (CTL) mediated killing. Irradiation induced a dose-dependent G2/M arrest in both cell lines irrespective from the irradiation source applied. Likewise, surface expression of the immunomodulatory molecules PD-L1, CD73, H2-Db and H2-Kb was increased in a dose-dependent manner. Both radiation modalities enhanced the susceptibility of tumor cells to CTL lysis, which was more pronounced in EO771/Luci/OVA cells than in PDA30364/OVA cells. Overall, compared to photon radiation, the effects of carbon ion radiation appeared to be enhanced at higher dose range for EO771 cells and extenuated at lower dose range for PDA30364/OVA cells. Our data show for the first time that equivalent doses of carbon ion and photon irradiation exert similar immunomodulating effects on the cell lines of both tumor entities, highlighted by an enhanced susceptibility to CTL mediated cytolysis in vitro.

Ku80 autoantigen as a cellular coreceptor for human parvovirus B19 infection

Blood ◽  
2005 ◽  
Vol 106 (10) ◽  
pp. 3449-3456 ◽  
Author(s):  
Yasuhiko Munakata ◽  
Takako Saito-Ito ◽  
Keiko Kumura-Ishii ◽  
Jie Huang ◽  
Takao Kodera ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Surface ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Parvovirus B19 ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Human Parvovirus B19 ◽  
Erythroid Cells ◽  
Interfering Rna

AbstractHuman parvovirus B19 (B19) infects human erythroid cells expressing P antigen. However, some cell lines that were positive for P antigen failed to bind B19, whereas some cell lines had an ability to bind B19 despite undetectable expression of P antigen. We here demonstrate that B19 specifically binds with Ku80 autoantigen on the cell surface. Furthermore, transfection of HeLa cells with the gene of Ku80 enabled the binding of B19 and allowed its entry into cells. Moreover, reduction of cell-surface expression of Ku80 in KU812Ep6 cells, which was a high-sensitive cell line for B19 infection, by short interfering RNA for Ku80 resulted in the marked inhibition of B19 binding in KU812Ep6 cells. Although Ku80 originally has been described as a nuclear protein, human bone marrow erythroid cells with glycophorin A or CD36, B cells with CD20, or T cells with CD3 were all positive for cell-surface expression of Ku80. B19 infection of KU812Ep6 cells and bone marrow cells was inhibited in the presence of anti-Ku80 antibody. Our data suggest that Ku80 functions as a novel coreceptor for B19 infection, and this finding may provide an explanation for the pathologic immunity associated with B19 infection.

scholarly journals The CXCR4–CXCL12-Axis Is of Prognostic Relevance in DLBCL and Its Antagonists Exert Pro-Apoptotic Effects In Vitro

2019 ◽  
Vol 20 (19) ◽  
pp. 4740 ◽  
Author(s):  
Katrin Pansy ◽  
Julia Feichtinger ◽  
Barbara Ehall ◽  
Barbara Uhl ◽  
Miriam Sedej ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Bone Marrow Involvement ◽  
B Cell Lymphoma ◽  
Cxcr4 Expression ◽  
Lymphoma Cell ◽  
Bone Marrow Biopsies ◽  
Cxcr4 Antagonists ◽  
Apoptotic Effects

In tumor cells of more than 20 different cancer types, the CXCR4-CXCL12-axis is involved in multiple key processes including proliferation, survival, migration, invasion, and metastasis. Since data on this axis in diffuse large B cell lymphoma (DLBCL) are inconsistent and limited, we comprehensively studied the CXCR4-CXCL12-axis in our DLBCL cohort as well as the effects of CXCR4 antagonists on lymphoma cell lines in vitro. In DLBCL, we observed a 140-fold higher CXCR4 expression compared to non-neoplastic controls, which was associated with poor clinical outcome. In corresponding bone marrow biopsies, we observed a correlation of CXCL12 expression and lymphoma infiltration rate as well as a reduction of CXCR4 expression in remission of bone marrow involvement after treatment. Additionally, we investigated the effects of three CXCR4 antagonists in vitro. Therefore, we used AMD3100 (Plerixafor), AMD070 (Mavorixafor), and WKI, the niacin derivative of AMD070, which we synthesized. WK1 demonstrated stronger pro-apoptotic effects than AMD070 in vitro and induced expression of pro-apoptotic genes of the BCL2-family in CXCR4-positive lymphoma cell lines. Finally, WK1 treatment resulted in the reduced expression of JNK-, ERK1/2- and NF-κB/BCR-target genes. These data indicate that the CXCR4-CXCL12-axis impacts the pathogenesis of DLBCL and represents a potential therapeutic target in aggressive lymphomas.

CXCR7 Regulates SDF-1 Induced Adhesion and Homing in Multiple Myeloma.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1674-1674 ◽  
Author(s):  
Nicholas Burwick ◽  
Anne-Sophie Moreau ◽  
Xiaoying Jia ◽  
Xavier Leleu ◽  
Judith Runnels ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Adhesion ◽  
Cell Lines ◽  
Cxcr4 Expression ◽  
Rt Pcr ◽  
Growth And Survival ◽  
Tumor Types

Abstract BACKGROUND: Multiple myeloma (MM) is a plasma cell malignancy that depends on interactions with the bone marrow (BM) microenvironment for growth and survival. In turn, adhesion of MM cells to the BM stroma provides a mechanism of resistance from standard chemotherapeutic agents. Recently, our lab has shown that by disrupting this adhesion using a selective CXCR4 inhibitor named AMD3100, MM cells are more sensitive to the proteasome inhibitor Bortezomib (Ghobrial lab, unpublished data). CXCR4 has been a particularly attractive target because its ligand SDF-1 is known to induce p42/44 MAPK, AKT, and the down-stream anti-apoptotic protein bad in MM cells, leading to increased MM growth and survival. Until recently, CXCR4 was thought to be a canonical receptor for the SDF-1 ligand. However, a second chemokine receptor for SDF-1 was subsequently discovered and named CXCR7. CXCR7 is a novel chemokine receptor that is important in cell adhesion, growth and survival in several tumor types. However, the role of CXCR7 in multiple myeloma (MM) has yet to be explored. Furthermore, the ability of SDF-1 ligand to regulate MM function via CXCR7 has not been studied. METHODS: The MM cell lines (U266, MM1.S, RPMI, OPM2, OPM1) were used. After informed consent was obtained, primary bone marrow samples from MM patients were collected. CD138 positive mononuclear cells were isolated by microbead selection. The expression of CXCR7 on MM cell lines and patient samples was confirmed using flow cytometry and RT-PCR analysis. For functional in vitro and ex-vivo assays, the CXCR7 selective antagonist 733 was used (ChemoCentryx Inc., Mountain View, CA). RESULTS: Here we show that CXCR7 was expressed on all tested MM cell lines and primary patient samples as demonstrated by flow cytometry and RT-PCR. Furthermore, CXCR7 was found to regulate SDF-1 induced MM cell adhesion, as demonstrated by in vitro assays using a small molecule compound specific for CXCR7 (733). The CXCR7 antagonist showed significant inhibition of adhesion of MM cell lines and patient samples to fibronectin, endothelial cells and stromal cells, with 50% reduction of adhesion at 5nM of the CXCR7 inhibitor, and with similar activity compared to 20uM of AMD3100 (CXCR4 inhibitor). However, unlike CXCR4, CXCR7 did not effect trans-well migration to SDF-1 chemokine. Interestingly, both receptors were found to be important for trans-endothelial migration of MM cells. Moreover, pre-treatment with 733 reduced homing of MM cells to the BM niche in vivo. Previous studies have failed to show signaling in response to CXCR7 in many tumor types. Here, we demonstrate that treatment with 733 inhibited SDF-1 induced pERK and pAKT, ribosomal pS6Kinase, pGSK3, pSTAT3, pFAK and pPAK signaling pathways, confirming a role for CXCR7 in facilitating SDF-1 signaling. This effect was further confirmed using immunofluorescence. To investigate whether CXCR7 and CXCR4 interact directly, we examined the effect of 733 and AMD3100 on CXCR4 expression and found that AMD3100 significantly inhibited CXCR4 expression, while 733 had no effect on CXCR4 expression, even in the presence of SDF-1. The CXCR7 inhibitor had no effect on the survival of MM cells using MTT and flow cytometry analysis, while high doses of 733 (1uM) had modest inhibition of proliferation. Interestingly, 733 prevented the growth advantage induced by 30nM SDF-1 at 24 hrs. CONCLUSION: Together, these results demonstrate the importance of CXCR7 in regulating MM adhesion and homing, and highlight the differential effects of CXCR4 and CXCR7 in regulating SDF-1 signaling in MM, thus providing a rationale for targeting the SDF-1/CXCR7 axis in MM.

Bone Marrow Interaction in Multiple Myeloma Pathogenesis: Phenotypical Analysis, Kinetics and Novel Therapy Approaches Based On CXCR4 Inhibition.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2450-2450
Author(s):  
Johannes Waldschmidt ◽  
Dagmar Wider ◽  
Marie Follo ◽  
Josefina Udi ◽  
Martina Kleber ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Adhesion Molecules ◽  
Cell Lines ◽  
Cxcr4 Expression ◽  
Cell Trafficking ◽  
Newly Diagnosed ◽  
Infiltration Rates ◽  
Dependent Cell ◽  
And Migration

Abstract Abstract 2450 Introduction: The interaction between malignant plasma cells and their microenvironment is central in multiple myeloma (MM) pathogenesis. Binding of MM cells to bone marrow (BM) stroma cells triggers the expression of adhesion molecules and secretion of chemo- and cytokines, promoting MM cell growth, drug resistance and migration. Stromal-derived factor-1 (SDF-1) and its receptor CXCR4 are essential for normal hematopoietic progenitor cell movement and adherence within the BM microenvironment. In leukemia and lymphoma, oncoproteins may inhibit SDF-1-dependent cell trafficking within the BM through a mechanism that is not fully understood. For that reason, understanding SDF-1-dependent cell trafficking within the BM and targeting MM-cell - host-BM interactions display a promising approach for the development of novel therapeutic strategies. Methods: BM samples of MM patients (n=59) were analysed using flow cytometry and compared to MGUS patients (n=3) and healthy volunteers (n=7). We compared patient samples with low BM infiltration (≤5%; n=13) intermediate (5–30%; n=29) and high infiltration rates (≥30%; n=17). We also assessed expression of adhesion molecules in MM patients with long-term disease control (n=20) vs. both newly diagnosed (n=16) and symptomatic MM patients (n=23) as previously grouped by San Miguel et al. (Haematologica July 6,2012). We also sought to elucidate in vitro, whether specific anti-MM agents (bortezomib, vorinostat, pomalidomide, EGCG), with and without M210B4 stroma support, and with and without the CXCR4 inhibitor AMD3100, target the interaction of MM cells. Experiments were performed using MM cell lines (U266, RPMI8226, L363, NCI-H929), the control T-cell line MOLT-4 and MM-patient BM samples. Cell viability was assessed via Trypan Blue- and AnnexinV/PI-staining. CD138, CXCR4 (SDF1-receptor), CD49d (VLA-4), CD11a (LFA-1) and CD44 (HERMES antigen) expression was evaluated by flow cytometry and ScanR microscopy. Results: In BM samples of MM patients as compared to MGUS and healthy volunteers, the CXCR4/CD138- (p=.036), CD49d/CD138- (p=.0013) and CD44/CD138-expression (p=.0072) was significantly amplified and correlated with increasing BM infiltration rates (p=.001). Both newly diagnosed and symptomatic MM patients confirmed significantly increased CXCR4/CD138-, CD49d/CD138- (p=.0013) and CD44/CD138-expression as compared to patients with long-term disease control. Of note, in newly diagnosed patients, the expression of adhesion molecules was even more enhanced than in symptomatic myeloma patients, underlining their critical and future potential role as targets for novel therapeutics. Comparison of MM cell lines' adhesion and migration markers with that of MM-patient BM specimens revealed U266 as the cell line most closely resembling human specimens. Cytotoxic effects with use of MM cell lines and bortezomib, vorinostat and pomalidomide confirmed prior cytotoxic concentrations. Cocultivation with stroma substantially reduced apoptosis and induced tumor protective effects. Additional AMD3100 treatment restored sensitivity to bortezomib, vorinostat and pomalidomide. CXCR4 expression was substantially reduced after AMD3100 treatment, while that of CD49d, CD44 and CD11a remained widely unchanged. Toxic or therapeutic effects of AMD3100 monotherapy were excluded for used doses of 50μM. Additional use of ScanR microscopy visualized co-localisation of CXCR4 expression both on the cell surface and within the cytoplasm of MM cells. ScanR microscopy results correlated with flow cytometry-determined CXCR4 expression. Ongoing analyses of both ScanR microscopy and flow cytometry will allow the detailed assessment of treatment studies with and without anti-MM agents and AMD3100. Conclusions: Our findings underline the critical role of adhesion and migration molecules in MM and may pave the way for novel therapeutic approaches targeting these microenvironmental mediators. Disclosures: No relevant conflicts of interest to declare.

The Novel CXCR4 Antagonist POL5551 Decreases Surface CXCR4 (s-CXCR4) Expression, Inhibits Chemotaxis, and Enhances Chemosensitivity in Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 780-780
Author(s):  
Edward Allan R. Sison ◽  
Daniel Magoon ◽  
Eric Chevalier ◽  
Klaus Dembowsky ◽  
Patrick Brown
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Cxcr4 Expression ◽  
Leukemia Cells ◽  
The Novel ◽  
Cxcr4 Antagonist ◽  
Induced Apoptosis

Abstract Abstract 780 Background: The interaction between the cell surface receptor CXCR4 and the chemokine SDF-1 (CXCL12) is critical in signaling between leukemic blasts and the bone marrow microenvironment. We previously demonstrated that CXCR4 is an important mediator of chemotherapy resistance, as chemotherapy-induced upregulation of s-CXCR4 in acute myeloid leukemia (AML) cell lines and primary samples led to increased SDF-1-mediated chemotaxis and increased protection by normal human bone marrow stroma from chemotherapy-induced apoptosis. We also showed that stromal protection and chemotherapy resistance could be reversed by treatment with the FDA-approved CXCR4 inhibitor plerixafor, both in vitro in stromal co-cultures of pre-B cell ALL cell lines and in vivo in xenografts of primary samples of infant MLL-rearranged ALL. Therefore, disruption of the CXCR4/SDF-1 axis is a rational means to target extrinsic survival mechanisms in acute leukemia. The novel Protein Epitope Mimetic (PEM) POL5551 is a selective and potent antagonist of CXCR4. Treatment with POL5551 inhibits vascular accumulation of CXCR4+ smooth muscle cells but its effects on ALL have not been reported. We hypothesized that treatment of ALL cell lines with POL5551 would 1) decrease s-CXCR4 expression, 2) inhibit SDF-1-mediated chemotaxis, and 3) reverse stromal-mediated protection from chemotherapy-induced apoptosis. Methods/Results: Pre-B cell ALL (697, HB11;19, NALM-6, SEMK2) and T cell ALL cell lines (CCRF-CEM-1301, Jurkat, Molt-4) were treated with dose ranges of POL5551. Cells were harvested at multiple time points over 72 hours and s-CXCR4 was measured by FACS. S-CXCR4 was potently and markedly reduced in all cell lines, with IC50 levels of <5 nM at 1 hour and IC50 levels of <20 nM at 48 hours. In comparison, 3- to 30-fold higher doses of plerixafor were needed to achieve similar levels of reduction. Simultaneous measurement of cell proliferation using the WST-1 proliferation assay demonstrated that treatment with POL5551 neither increased nor decreased leukemia cell proliferation in a significant manner. To ascertain the functionality of s-CXCR4 inhibition, we performed chemotaxis assays. Leukemia cells were treated with 10 nM POL5551 or vehicle control and placed into hanging cell culture inserts. Migration through a permeable membrane toward an SDF-1 gradient was then measured after 24 hours. Compared to control-treated cells, POL5551-treated cells had significantly decreased SDF-1-induced chemotaxis (average 38% reduction in chemotaxis in pre-B cell lines, p<0.001; average 41% reduction in T cell lines, p=0.05). We also performed co-culture experiments with normal human bone marrow stroma in the presence and absence of POL5551 to further demonstrate the functional effects of s-CXCR4 inhibition. Specifically, we cultured leukemia cells off stroma (O), on stroma (S), or pretreated with POL5551 for 30 minutes prior to plating on stroma (P+S). Cells from each culture condition were then treated with dose ranges of chemotherapy. Following treatment, we measured apoptosis by staining with Annexin V/7-AAD. IC10 through IC90 values were obtained using Calcusyn. To quantify stromal protection, we calculated a Protective Index (PI), defined as the S IC values divided by the O IC values. Thus, PI >1 signified stromal protection, while PI ≤1 signified no stromal protection. To quantify the ability of POL5551 to reverse stromal protection, we calculated a Reversal Index (RI), defined as the P+S IC values divided by the O IC values. Therefore, PI > RI indicated a decrease in stromal protection, while RI ≤1 indicated a reversal of stromal protection. Overall, stroma protected leukemia cells from chemotherapy-induced apoptosis. Importantly, treatment with POL5551 abrogated stromal-mediated protection and restored chemosensitivity (eg, PI 1.182 vs. RI 0.956 for NALM-6 treated with daunorubicin +/− 20 nM POL5551, p<1×10e-9). Conclusions: The novel CXCR4 antagonist POL5551 is a potent inhibitor of CXCR4 in pre-B and T ALL cell lines with activity at nanomolar concentrations in decreasing s-CXCR4 expression, inhibiting SDF-1-induced chemotaxis, and reversing stromal-mediated protection from chemotherapy in vitro. Therefore, if our findings are confirmed in primary samples and in vivo, interruption of leukemia-microenvironment signaling with POL5551 may prove to be an effective strategy in the treatment of pre-B and T cell ALL. Disclosures: Chevalier: Polyphor Ltd: Employment. Dembowsky:Polyphor Ltd: Employment.

Inhibition Of PI3K Classia Kinases Using GDC0941 Overcomes Protection Of Multiple Myeloma Cells In The Bone Marrow Microenvironment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3169-3169
Author(s):  
Hugh Kikuchi ◽  
Amofa Eunice ◽  
Maeve McEnery ◽  
Farzin Farzaneh ◽  
Stephen A Schey ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Bone Resorption ◽  
Cell Lines ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Pi3k Pathway ◽  
Dependent Manner ◽  
Dose Dependent

Abstract Despite of newly developed and more efficacious therapies, multiple myeloma (MM) remains incurable as most patient will eventually relapse and become refractory. The bone marrow (BM) microenvironment provides niches that are advantageous for drug resistance. Effective therapies against MM should ideally target the various protective BM niches that promote MM cell survival and relapse. In addition to stromal mesenchymal/myofibroblastic cells, osteoclasts play a key supportive role in MM cell viability. Additionally, 80% of patients develop osteolytic lesions, which is a major cause of morbidity. Increased osteoclast activity is characteristic in these patients and targeting osteoclast function is desirable to improve therapies against MM. Osteoclasts need to form an F-actin containing ring along the cell margin that defines a resorbing compartment where protons and degradative enzymes are secreted for dissolution of bone mineral. Remodelling of F-actin and vesicle secretion are regulated by the class IA PI3K pathway during osteoclastic bone resorption. Additionally, it has recently been shown that inhibition of the class IA PI3K pathway in MM cells with GDC0941 induces apoptosis-mediated killing. We hypothesised that GDC0941 could be used as a therapeutic agent to overcome MM-induced osteoclast activation. GDC0941 inhibited maturation of osteoclasts derived from BM aspirates from MM patients in a dose dependent manner. This correlated with decreased bone resorption of osteoclasts cultured on dentine discs. Exposure of mature osteoclasts to GC0941 resulted in abnormal organisation of larger F-actin rings, suggesting a negative effect on the dynamics of the actin cytoskeleton required for bone resorption. We also found that GDC-0941 can prevent protection of the MM cell lines MM1.S and MM1.R by osteoclasts against killing. GDC-0941 alone blocked MM cell proliferation independently of the presence of BM stromal cells and synergised with other therapeutic agents including Lenalidomide, Pomalidomide, Bortezomid and Dexamethasone. We also found that in the presence of MM cells, Dexamethasone (a drug commonly used alone or in combination with new drugs against MM) induced the proliferation of BM stromal cells and adhesion of MM cells on this protective stroma in a dose dependent manner. Dexamethasone is highly effective at MM cell killing when cells are cultured alone. However, we found that at low doses (below 1 uM) and in the presence of BM stromal cells, Dexamethasone could induce MM cell proliferation. GDC0941 enhanced Dexamethasone killing even in the presence of BM stromal cells by blocking Dexamethasone-induced stromal cell proliferation and adhesion of MM cells on the stroma. Targeting individual the PI3K Class IA isoforms alpha, beta, delta or gamma proved to be a less efficient strategy to enhance Dexamethasone killing. Previous work has shown that efficacy of targeting individual PI3K Class I A isoforms would be low for activation of caspases in MM cells as it would be dependent on relative amounts of isoforms expressed by the MM patient. GDC-0941 also inhibited the proliferation of MM1.R and RPMI8266 MM cell lines, which are less sensitive to treatment to Dexamethasone. Co-culture of MM cells with BM stromal cells induced the secretion of IL-10, IL-6, IL-8, MCP-1 and MIP1-alpha. The dose-dependant increased proliferation of Dexamethasone-treated MM cells in the presence of the BM stroma correlated with the pattern of secretion of IL-10 (a cytokine that can induce B-cell proliferation) and this was blocked by the combination of Dexamethasone with GDC0941. GDC-0941 alone or in combination with Dexamethasone was more efficacious at inducing MM cell apoptosis in the presence of the BM stroma cells vs treatment of MM cells alone. These are very encouraging results as they suggest that GDC-0941 in combination with Dexamethasone would be potentially highly efficacious for targeting MM cells in the BM microenvironment. We are currently performing in vivo data using C57BL/KaLwRij mice injected with 5T33-eGFP MM cells that will be discussed at the meeting. We propose that MM patients with active bony disease may benefit from treatment with GDC0941 alone or in combination with currently used therapeutic drugs against MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Role for Syntenin-1 in Multiple Myeloma Cell Survival

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1008-1008
Author(s):  
Tyler Moser-Katz ◽  
Catherine M. Gavile ◽  
Benjamin G Barwick ◽  
Sagar Lonial ◽  
Lawrence H. Boise
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Death ◽  
Cell Survival ◽  
Cell Lines ◽  
Plasma Cells ◽  
Surface Expression ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Rpmi 8226

Abstract Multiple myeloma is the second most common hematological malignancy in the U.S. with an estimated 30,700 new diagnoses in 2018. It is a clonal disease of plasma cells that, despite recent therapeutic advances, remains incurable. Myeloma cells retain numerous characteristics of normal plasma cells including reliance on survival signals in the bone marrow for long term viability. However, malignant transformation of plasma cells imparts the ability to proliferate, causing harmful bone lesions in patients, and in advanced stages independence of the bone-marrow microenvironment. Therefore, we are investigating the molecular mechanisms of myeloma cell survival that allow them to become extramedullary. We identified syntenin-1 (SDCBP) as a protein involved in myeloma cell survival and a potential therapeutic target. Syntenin-1 is an adapter protein that has been shown to regulate surface expression of several transmembrane proteins by binding with membrane phospholipids and mediating vesicular trafficking of proteins throughout the cell. Syntenin-1 regulates the surface expression of CD138, a plasma/myeloma cell marker. Syntenin-1 has been shown to regulate apoptosis in numerous cancer cell lines including breast cancer, glioma, and pancreatic cancer but its role in multiple myeloma survival has not been studied. To determine if syntenin-1 expression has an effect on myeloma cell survival, we utilized the CoMMpass dataset (IA12), a longitudinal study of myeloma patients that includes transcriptomic analysis throughout treatment. We found that patients with the highest expression of syntenin-1 mRNA (top quartile) had significantly worse overall survival, progression-free survival, and a shorter response duration than those in the bottom quartile of expression. To determine if syntenin-1 has a role in myeloma cell survival, we used short hairpin RNA to knock down syntenin-1 (shsyn) in RPMI 8226 and MM1.s myeloma cell lines. We then determined the amount of cell death using Annexin-V staining flow cytometry four days following lentiviral infection. We found increased cell death in syntenin-1-silenced cells compared to our empty vector control in both RPMI 8226 (control=42.17%, shsyn=71.53%, p=0.04) and MM1.s cell lines (control=8.57%, shsyn=29.9%, p=0.04) suggesting that syntenin-1 is important for myeloma cell survival. Syntenin-1 contains two PDZ domains that allow it to bind to receptor proteins via their corresponding PDZ-binding motifs. We therefore wanted to look at correlation of syntenin-1 expression with CD138 and CD86, two PDZ-binding domain containing proteins expressed on the surface of myeloma cells. Using the CoMMpass dataset, we found patients with high expression of syntenin-1 had a median expression of CD86 that was twice as high as the total population (P<0.0001) while syntenin-1-low patients expressed CD86 at levels that were half as much as the population (P<0.0001). In contrast, there was no clear relationship between syntenin-1 and CD138 mRNA expression. Indeed if one takes into account all patients, there is a positive correlation between CD86 and syntenin-1 expression (r=0.228, P<0.0001) while there is a negative correlation between CD138 and syntenin-1 (r=-0.1923, P<0.0001). The correlation with CD86 but not CD138 suggests a previously undescribed role for syntenin-1 in myeloma cells. Our lab has previously shown that expression of CD86 is necessary for myeloma cell survival, and signals via its cytoplasmic domain to confer drug resistance. Silencing syntenin-1 results in a decrease in CD86 surface expression. However, there is no change in CD86 transcript or total cellular CD86 protein levels in our shsyn treated cells. Moreover, knockdown of CD86 resulted in increased protein expression and transcript levels of syntenin-1. Taken together, these data suggest that syntenin-1 may regulate CD86 expression on the cell surface. Our data supports a novel role for syntenin-1 in myeloma cell viability and as a potential regulator of CD86 surface expression. The role of syntenin-1 has not previously been explored in multiple myeloma and determining its molecular function is warranted as it may be an attractive target for therapeutic treatment of the disease. Disclosures Lonial: Amgen: Research Funding. Boise:AstraZeneca: Honoraria; Abbvie: Consultancy.

Comparison of CXCR-4 and Adhesion Molecule Expression in Healthy Bone Marrow with Expression in Bone Marrow and Peripheral Blood of Patients Receiving G-CSF Plus AMD3100.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1974-1974
Author(s):  
Uta Oelschlaegel ◽  
Martin Bornhaeuser ◽  
Frank Kroschinsky ◽  
Gerhard Ehninger ◽  
Uwe Platzbecker
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Adhesion Molecules ◽  
Peripheral Blood ◽  
Surface Expression ◽  
Stem Cell Mobilization ◽  
Qualitative And Quantitative ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
Cell Mobilization

Abstract It is known that the crosstalk between adhesion molecules, bone marrow microenvironment, and cytokines facilitates the multi step process of stem cell mobilization from bone marrow to peripheral blood. A combination of G-CSF plus AMD3100 - a CXCR-4 antagonist - has been shown to be safe and efficient in stem cell mobilization of healthy donors and cancer patients. Nevertheless, data predicting the efficacy of this approach are still missing. The present study investigated the correlation of the expression of CXCR-4 (CD184) and adhesion molecules with the kinetics and efficacy of stem cell mobilization in nine patients with Multiple Myeloma (MM) or NHL, respectively. Steady-state mobilization was performed using a combination of G-CSF (Filgrastim, 10μg/kg/d, 8 am) for 4 days followed by AMD3100 (240μg/kg) on day 4 at 10pm. Autologous aphereses were started on day 5. Bone marrow and peripheral blood (PB) before AMD3100 application (day 4) and PB on day 5 were investigated with a 4-color flow cytometric procedure. Bone marrow aspirates of healthy donors (n=20) served as control. The qualitative (%) and quantitative (mean fluorescence intensity, [MFI]) antigen expression of CXCR-4 in relation to CD34 was assessed as well as the expression of certain adhesion molecules including LFA-1, PECAM-1, VLA-1, L-selectin and CD44. First, the median percentage of CXCR-4 surface expression in healthy bone marrow was significantly higher (92%; range: 52 – 99%) than in patients bone marrow (70%; 30 – 88%; p=0.002), PB before AMD3100 (87%; 35 – 97%; p=0.050) and on day 5 (17%; 2 – 74%; p<0.001), whereas cytoplasmic expression was comparable (91%; 53 – 95%) in all cell compartments. The median quantitative CXCR-4 surface expression was significantly decreased in PB on day 5 compared to pre AMD3100 (14 vs. 95; p=0.003). Furthermore, the qualitative expression of LFA-1 and the quantitative expression of LFA-1, PECAM-1, VLA-1, and CD44 were also downregulated in response to AMD3100 (p<0.010). Second, a median of 63/μl (range: 15 – 132/μl) CD34+ cells was measured in the PB on day 5. Thus, a high absolute count of CD34+ cells in the PB on day 5 significantly correlated with lower qualitative and quantitative CXCR-4 expression in the same material (r=0.833; p=0.015). Evaluating CXCR-4 expression in bone marrow, PB before AMD3100 and on day 5 no significant correlation to CD34+ counts could be detected. However, there was one very poor mobilizing patient (15/μl CD34+ cells on day 5) in whom the quantitative CXCR-4 expression in the bone marrow was significantly higher than the median of all patients (MFI 95 vs. 26). Furthermore, some of the adhesion molecules (L-selectin, VLA-4, and CD44) showed a rather positive correlation with CD34 count. In summary, these preliminary data suggest that the amount of CD34+ cells in the peripheral blood after G-CSF plus AMD3100 application seems to be negatively correlated with CXCR-4 expression. A higher quantitative CXCR-4 expression in the bone marrow pre AMD3100 might predict a lower mobilization efficacy.

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