Surface CXCR4 Research Articles

Combined Targeting of CXCR2 and VLA4 Results in Rapid and Synergistic Mobilization of Hematopoietic Stem and Progenitor Cells in Mice

Introduction:Since the first description of hematopoietic stem and progenitor cell (HSPC) mobilization over forty years ago, it has become the standard of care for both autologous and allogeneic transplantation. A five-day course of G-CSF represents the most commonly used mobilization regimen today. The CXCR4 inhibitor, plerixafor, is a more rapid but weak mobilizer when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects.Methods:Given the critical role of VLA4/VCAM1 signaling for migration and retention of HSPC, we were seeking to identify small molecule antagonists of VLA4 with improved potency and bioavailability. Relative to previously described comparators Bio5192 (□4□1-specific) and firategrast (□4□1 and □4□7 dual-specific), our lead candidate, CWHM-823, exhibited increased aqueous solubility and ~10-100 fold better activity in blocking VLA4 and mobilizing HSPC in mice. CWHM-823 pharmacokinetics and mobilization were assessed in BALB/c and DBA/2 mice at different doses (3 to 15 mg/kg) and time points (15 to 240 min) when administered alone or in combination with the truncated isoform of the CXCR2 agonist Gro-beta (tGro-β, 2.5 mg/kg, generously provided by GlaxoSmithKline). HSPC mobilization was monitored using flow cytometry and clonogenic in vitro assays. "True" stem cells were measured in a serial competitive transplantation assay. The combination of tGro-β and VLA4 antagonist was further tested in diabetic mice in comparison to G-CSF (9 x 100μg/kg, q12h). RNA profiling of flow-sorted HSPC was performed via microarray analysis.Results:The combination of tGro-β with each VLA4 antagonist resulted in a dramatic synergistic increase in circulating HSPC numbers when compared to steady state (50-70-fold) or treatment with single agents (3-10 fold) including tGroβ. Mobilization with tGro-β + CWHM-823 was rapid, peaking at 15-30 minutes after injection. In a model of streptozotocin-induced diabetes, the mobilopathy (reduction in stem cell mobilization compared to wild type mice) was considerably less pronounced with the combination tGro-β + CWHM-823 (~1.5-fold lower CFU mobilization in diabetic mice) versus the 5-day course of G-CSF (~3-fold reduction).Despite the superior progenitor cell mobilization achieved with G-CSF (~2-fold more CFU and LSK/ml), the concentration of serially repopulating units (RU) was equally high in the tGro-β + CWHM-823 and G-CSF mobilized grafts suggesting a higher HSC frequency (1 RU out of 200 vs. 1 RU out of 400 LSK/CFU) in the tGro-β + CWHM-823 mobilized grafts (Figure 1). RNA profiling demonstrated close similarity between the expression profile of tGro-β + CWHM-823 mobilized, BM resident, and G-CSF mobilized LSK, with less than 0.5% of genes found to be significantly up- or downregulated.CXCR2 chemokine receptor stimulation was critical for the observed synergistic response, as pretreatment ("priming") or simultaneous treatment with tGro-β resulted in subsequent enhanced mobilization using VLA4 inhibitors, whereas reversed administration (VLA4 antagonist followed by tGro-β) had no effect on potency of either agent. Lack of surface CXCR2 expression on HSPC suggested that a rapidly acting effector molecule released from tGro-β-stimulated mature myeloid cells may subsequently influence VLA4-mediated HSPC adhesion/retention. Consistent with this theory, we observed increased protease MMP-9 in plasma within minutes after treatment with tGro-β + CWHM-823.Conclusions:We describe a novel strategy for rapid, reliable, and potent mobilization of HSPC in mice using a combination of VLA4 blockade (via novel and potent □4□1 inhibitors) and CXCR2 activation (via tGro-β). The combination of tGro-β + VLA4 inhibitors or tGro-β followed by VLA4 inhibitors results in synergistic and rapid HSPC mobilization with quantity and quality of repopulating units similar to optimal mobilization with G-CSF. These data suggest further development of tGro-β + VLA4 inhibitor combinations for clinical testing is warranted. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Attenuation of chemokine receptor function and surface expression as an immunomodulatory strategy employed by human cytomegalovirus is linked to vGPCR US28.

BackgroundSome herpesviruses like human cytomegalovirus (HCMV) encode viral G protein-coupled receptors that cause reprogramming of cell signaling to facilitate dissemination of the virus, prevent immune surveillance and establish life-long latency. Human GPCRs are known to function in complex signaling networks involving direct physical interactions as well as indirect crosstalk of orthogonal signaling networks. The human chemokine receptor CXCR4 is expressed on hematopoietic stem cells, leukocytes, endothelial and epithelial cells, which are infected by HCMV or display reservoirs of latency.ResultsWe investigated the potential heteromerization of US28 with CXCR4 as well as the influence of US28 on CXCR4 signaling. Using Bioluminescence Resonance Energy Transfer and luciferase-complementation based methods we show that US28 expression exhibits negative effects on CXCR4 signaling and constitutive surface expression in HEK293T cells. Furthermore, we demonstrate that this effect is not mediated by receptor heteromerization but via signaling crosstalk. Additionally, we show that in HCMV, strain TB40E, infected HUVEC the surface expression of CXCR4 is strongly downregulated, whereas in TB40E-delUS28 infected cells, CXCR4 surface expression is not altered in particular at late time points of infection.ConclusionsWe show that the vGPCR US28 is leading to severely disturbed signaling and surface expression of the chemokine receptor CXCR4 thereby representing an effective mechanism used by vGPCRs to reprogram host cell signaling. In contrast to other studies, we demonstrate that these effects are not mediated via heteromerization.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-016-0154-x) contains supplementary material, which is available to authorized users.

Neuropilin-1 Is an Important Niche Component and Exerts Context-Dependent Effects on Hematopoietic Stem Cells.

Marrow adipocytes pose a significant problem in post-transplant regeneration of hematopoiesis owing to their negative effects on regeneration of hematopoiesis. However, the precise mechanism operative in this negative regulation is not clear. In this study, we show that marrow adipocytes express neuropilin-1 (NRP1) as a function of differentiation and inhibit regeneration of hematopoiesis by three principal mechanisms: one, by inducing apoptosis in hematopoietic stem/progenitor cells (HSPCs) through the death receptor-mediated pathway; two, by downregulating CXCR4 expression on the HSPCs through ligand-mediated internalization; and three, by secreting copious amounts of transforming growth factor β1 (TGFβ1), a known inhibitor of hematopoiesis. Silencing of NRP1 in these adipocytes rescued the apoptosis of cocultured HSPCs and boosted the CXCR4 surface expression on them, showing an active role of NRP1 in these processes. However, such silencing had no effect on TGFβ1 secretion and consequent inhibition of hematopoiesis by them, showing that secretion of TGFβ1 by adipocytes is independent of NRP1 expression by them. Surprisingly, mesenchymal stromal cells modified with NRP1 supported expansion of HSPCs having enhanced functionality, suggesting that NRP1 exerts a context-dependent effect on hematopoiesis. Our data demonstrate that NRP1 is an important niche component and exerts context-dependent effects on HSPCs. Based on these data, we speculate that antibody- or peptide-mediated blocking of NRP1-HSC interactions coupled with a pharmacological inhibition of TGFβ1 signaling may help in combating the negative regulation of post-transplant regeneration of hematopoiesis in a more effective manner.

Expansion and Productive HIV-1 Infection of Foxp3 Positive CD4 T Cells at Pleural Sites of Hiv/Tb Co-Infection

CD4 T-cells expressing Foxp3 are expanded systemically during active tuberculosis (TB) regardless of HIV-1 co-infection. Foxp3+ CD4 T cells are targets of HIV-1 infection. However, expansion of HIV-1 infected Foxp3+ CD4 T cells at sites of HIV/TB co-infection, and whether they contribute to promotion of HIV-1 viral activity is not known. Pleural fluid mononuclear cells (PFMC) from HIV/TB co-infected patients with pleural TB were characterized by immune-staining and FACS analysis for surface markers CD4, CD127, CCR5, CXCR4, HLA-DR and intracellular expression of Foxp3, HIVp24, IFN-γ and Bcl-2. Whole PFMC and bead separated CD4+CD25+CD127- T cells were assessed for HIV-1 LTR strong stop (SS) DNA by real-time PCR, which represents viral DNA post cell entry and initiation of reverse transcription. High numbers of HIV-1 p24 positive Foxp3+ and Foxp3+CD127- CD4 T cells were identified in PFMC from HIV/TB co-infected subjects. CD4+Foxp3+CD127- T cells displayed high expression of the cellular activation marker, HLA-DR. Further, expression of the HIV-1 co-receptors, CCR5 and CXCR4, were higher on CD4+Foxp3+T cells compared to CD4+Foxp3- T cells. Purified CD4+CD25+CD127- T cells isolated from PFMC of HIV/TB co-infected patients, were over 90% CD4+Foxp3+T cells, and exhibited higher HIV-1 SS DNA as compared to whole PFMC, and as compared to CD4+CD25+CD127- T cells from an HIV-infected subject with pleural mesothelioma. HIV-1 p24+ Foxp3+ CD4+T cells from HIV/TB patients higher in Bcl-2 expression as compared to both HIV-1 p24+ Foxp3- CD4 T cells, and Foxp3+ CD4+T cells without HIV-p24 expression. Foxp3+ CD4 T cells in PFMC from HIV/TB co-infected subjects are predisposed to productive HIV-1 infection and have survival advantage as compared to Foxp3 negative CD4 T cells.

Milk fat globule-EGF factor 8 suppresses the aberrant immune response of systemic lupus erythematosus-derived neutrophils and associated tissue damage

Abnormal features of the systemic lupus erythematosus (SLE)-derived neutrophils, promoted aberrant immune response, have inspired new studies of the induction of autoimmunity and the development of organ damage in SLE. In this study, we explore the effect of milk fat globule-EGF factor 8 (MFG-E8) on the aberrant nitrification features in pristane-induced lupus. SLE patients and mice with pristane-induced lupus develop autoantibodies associated with MFG-E8 overproduction. However, the deletion of MFG-E8 leads to uncontrolled early pulmonary and peritoneal inflammation and tissue damage in mice with pristane-induced lupus. Consistent with these findings, MFG-E8-deficient mice that are exposed to pristane show enhanced neutrophil accumulation and increased neutrophil death, including apoptosis, necrosis and NETosis, as well as impaired phagocytosis of macrophages. The consequences are the expansion of diffuse pulmonary hemorrhage, increased anti-nuclear antibody, anti-dsDNA antibody and anti-neutrophil cytoplasmic antibody levels, and enhanced immune complexes deposition and neutrophil extracellular traps (NETs) formation in the lung and kidney tissues of MFG-E8-deficient mice exposed to pristane. In patients with SLE and mice with pristane-induced lupus, neutrophil accumulation is elevated, which depends on higher expression of the surface receptor CXCR2. After pretreatment with recombinant MFG-E8, the surface expression of CXCR2 on neutrophil is downregulated, and the MFG-E8 deletion increase CXCR2 expression by ~40%. These studies indicate that MFG-E8 reduces neutrophil migration and NETosis via downregulating surface CXCR2 expression in parallel with its role in the phagocytosis of apoptotic neutrophils, suggesting that MFG-E8 may serve as a therapeutic agent for attenuating the early inflammatory responses of SLE and protect patients from lupus-related damage.

Contribution and underlying mechanisms of CXCR4 overexpression in patients with systemic lupus erythematosus.

Aberrant expression of CXCR4 has been indicated to play a role in the pathogenesis of systemic lupus erythematosus (SLE), but the mechanism of CXCR4 dysregulation in SLE is unclear. This study is aimed to explore the clinical significance and possible mechanisms of abnormal CXCR4 expression on B cells from patients with untreated SLE. Expression of CXCR4 on peripheral B cells was determined by flow cytometry and western blotting. Freshly isolated B cells were cultured with exogenous interleukin 21(IL-21) in the presence or absence of CD40 ligand (CD40L) plus anti-IgM antibody (aIgM), and changes in CXCR4 expression were detected. Involvement of phosphatidylinositol 3 kinase (PI3K)/Akt and Janus kinase/Signal transducer and activator of transcription (JAK/STAT) signaling pathways was assessed by adding blocking agents Ly294002 and AG490. Since CD63 is reported to mediate endosomal recruitment of CXCR4 and BCL6 is capable of silencing CD63 gene transcription, we also measured BCL6 and CD63 gene transcription with real-time PCR. It was shown that CXCR4 expression on B cells was significantly upregulated in SLE patients, especially in those with lupus nephritis, and was positively correlated with SLE Disease Activity Index scores and negatively with the serum complement 3 levels (P<0.05). Downregulation of CXCR4 by IL-21 was intact. In contrast, a similar effect of aIgM plus CD40L in downregulating CXCR4 expression was defective in SLE patients but was restored by co-stimulation with IL-21 in vitro. Both Ly294002 and AG490 promoted downregulation of surface CXCR4 expression on B cells from SLE patients (P=0.078 and P=0.064). Furthermore, B cells from SLE patients exhibited diminished CD63 mRNA and enhanced BCL6 mRNA expression (both P<0.05). To sum up, CXCR4 was overexpressed on SLE B cells, positively correlating with disease activity and kidney involvement. Overactivation of the PI3K/Akt and JAK/STAT pathways as well as defective CD63 synthesis may contribute to CXCR4 dysregulation in SLE.

Abstract 1171: LIMK1 functions as a modulator of expression and targeting of CXCR4 in prostate cancer cells through a negative feedback loop

Abstract Increased activation of the GPCR CXCR4/CXCL12 signaling axis is commonly noted in metastatic prostate cancer and shows an association with poor prognosis. CXCR4, and its ligand, CXCL12/SDF-1a, are recognized as powerful mediators for tumor microenvironment remodeling and chemoprotection. LIM kinase 1 (LIMK1), a serine/threonine kinase, is an actin and microtubule modulatory protein that is also overexpressed in a variety of cancers including prostate cancer. Earlier, we showed that LIMK1 is involved in cell invasion and inhibition of LIMK1 expression reverted the invasive phenotype of PC3 prostate cancer cells. Activation of CXCR4/CXCL12 axis promotes activation of LIMK1 through RhoGTPAse pathway and promotes docetaxel resistance. Here we show that LIMK1 expression and functions have a positive correlation with CXCR4 expression and subcellular localization. We used metastatic prostate cancer cell lines PC3 and M12 and the non-tumorigenic prostate cell lines BPH and P69 to monitor expression and membrane localization of CXCR4 using western blots, flow cytometry and immunofluorescence analysis. Our analysis showed that cells with higher expression of LIMK1 (PC3) overexpress CXCR4, and that cell lines with low levels of LIMK1 (BPH and P69) express relatively lower amounts of CXCR4. Flow cytometric analysis showed a stronger surface staining of CXCR4 in the LIMK1 overexpressing PC3 cells compared to BPH cells. Next, we studied the effect of shRNA-mediated inhibition of LIMK1 expression on CXCR4 expression in PC3 or M12 cells. Western blot and immunofluorescence analysis showed noticeable reduction in CXCR4 levels, and its surface localization, as noted by flow cytometry, in LIMK1 shRNA transfected PC3 and M12 cells. To assess if the kinase activity of LIMK1 is essential for CXCR4 expression and surface localization, we treated these cells with LIMK1 specific kinase inhibitor BMS-5 and determined expression of CXCR4. Western blots and immunofluorescence analysis showed reduced expression of CXCR4 in treated cells compared to vehicle treated cells. Our observation indicates an indirect association between LIMK1 and CXCR4 expression and shows for the first time that expression of LIMK1 is required for the maintenance of CXCR4 expression. Our study demonstrates a novel positive feed back regulatory mechanism between LIMK1 and CXCR4 and suggest a possible combination treatment option through inhibition of LIMK1 expression and/or its kinase activity for improving chemosensitivity of prostate cancer cells with overactive CXCR4/CXCL12 axis. Citation Format: Richard Ottman, Lisa Ritchey, Ryan Herchan, Alexia Bossan, Ratna Chakrabarti. LIMK1 functions as a modulator of expression and targeting of CXCR4 in prostate cancer cells through a negative feedback loop. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1171.

Effects of short-term inflammatory and/or hypoxic pretreatments on periodontal ligament stem cells: in vitro and in vivo studies

In this study, we extensively screened the in vitro and in vivo effects of PDLSCs following short-term inflammatory and/or hypoxic pretreatments. We found that the 24-h hypoxic pretreatment of PDLSCs significantly enhanced cell migration and improved cell surface CXCR4 expression. In addition, hypoxia-pretreated PDLSCs exhibited improved cell colony formation and proliferation. Cells that were dually stimulated also formed more colonies compared to untreated cells but their proliferation did not increase. Importantly, the hypoxic pretreatment of PDLSCs enhanced cell differentiation as determined by elevated RUNX-2 and ALP protein expression. In this context, the inflammatory stimulus impaired cell OCN protein expression, while dual stimuli led to decreased RUNX-2 and OCN mRNA levels. Although preconditioning PDLSCs with inflammatory and/or hypoxic pretreatments resulted in no differences in the production of matrix proteins, hypoxic pretreatment led to the generation of thicker cell sheets; the inflammatory stimulus weakened the ability of cells to form sheets. All the resultant cell sheets exhibited clear bone regeneration following ectopic transplantation as well as in periodontal defect models; the amount of new bone formed by hypoxia-preconditioned cells was significantly greater than that formed by inflammatory stimulus- or dual-stimuli-treated cells or by nonpreconditioned cells. The regeneration of new cementum and periodontal ligaments was only identified in the hypoxia-stimulus and no-stimulus cell groups. Our findings suggest that PDLSCs that undergo short-term hypoxic pretreatment show improved cellular behavior in vitro and enhanced regenerative potential in vivo. The preconditioning of PDLSCs via combined treatments or an inflammatory stimulus requires further investigation.

The CXCR4 antagonist plerixafor enhances the effect of rituximab in diffuse large B-cell lymphoma cell lines.

BackgroundDiffuse large B-cell lymphoma (DLBCL) is an aggressive disease with variable clinical outcome, accounting for at least 25-30 % of adult non-Hodgkin lymphomas. Approximately one third of DLBCL patients are not cured by the currently used treatment regimen, R-CHOP. Hence, new treatment strategies are needed. Antagonizing the CXCR4 receptor might be promising since the CXCR4-CXCL12 axis is implicated in several aspects of tumor pathogenesis as well as in protection from chemotherapeutic response. In Burkitt lymphoma, the CXCR4 antagonist plerixafor has already been shown to enhance the therapeutic effect of rituximab, the immunotherapeutic agent of R-CHOP; but this is yet to be confirmed for DLBCL. We, therefore, investigated the effect of plerixafor on DLBCL cellular response to rituximab.MethodsIn this in vitro study, human DLBCL cell lines were treated with rituximab and/or plerixafor, concomitantly or in sequence. The trypan blue exclusion method and MTS-based assays were used to evaluate cellular proliferation, whereas flow cytometry was used for assessment of apoptosis status and CXCR4 surface expression level. Linear mixed effects models were used to assess statistical significance.ResultsWe observed that simultaneous addition of plerixafor and rituximab resulted in a significant decrease in DLBCL cellular proliferation, compared to monotherapeutic response. The effect was dose-dependent, and concomitant administration was observed to be superior to sequential drug administration. Accordingly, the fraction of apoptotic/dead cells significantly increased following addition of plerixafor to rituximab treatment. Furthermore, exposure of DLBCL cells to plerixafor resulted in a significant decrease in CXCR4 fluorescence intensity.ConclusionsBased on our results, implying that the anti-proliferative/pro-apoptotic effect of rituximab on DLBCL cells can be synergistically enhanced by the CXCR4 antagonist plerixafor, addition of plerixafor to the R-CHOP regimen can be suggested to improve treatment outcome for DLBCL patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s40364-016-0067-2) contains supplementary material, which is available to authorized users.

Targeted positron emission tomography imaging of CXCR4 expression in patients with acute myeloid leukemia.

Acute myeloid leukemia originates from leukemia-initiating cells that reside in the protective bone marrow niche. CXCR4/CXCL12 interaction is crucially involved in recruitment and retention of leukemia-initiating cells within this niche. Various drugs targeting this pathway have entered clinical trials. To evaluate CXCR4 imaging in acute myeloid leukemia, we first tested CXCR4 expression in patient-derived primary blasts. Flow cytometry revealed that high blast counts in patients with acute myeloid leukemia correlate with high CXCR4 expression. The wide range of CXCR4 surface expression in patients was reflected in cell lines of acute myeloid leukemia. Next, we evaluated the CXCR4-specific peptide Pentixafor by positron emission tomography imaging in mice harboring CXCR4 positive and CXCR4 negative leukemia xenografts, and in 10 patients with active disease. [(68)Ga]Pentixafor-positron emission tomography showed specific measurable disease in murine CXCR4 positive xenografts, but not when CXCR4 was knocked out with CRISPR/Cas9 gene editing. Five of 10 patients showed tracer uptake correlating well with leukemia infiltration assessed by magnetic resonance imaging. The mean maximal standard uptake value was significantly higher in visually CXCR4 positive patients compared to CXCR4 negative patients. In summary, in vivo molecular CXCR4 imaging by means of positron emission tomography is feasible in acute myeloid leukemia. These data provide a framework for future diagnostic and theranostic approaches targeting the CXCR4/CXCL12-defined leukemia-initiating cell niche.

Glucocorticoids Suppress CCR9-Mediated Chemotaxis, Calcium Flux, and Adhesion to MAdCAM-1 in Human T Cells.

CCR9 expressed on T lymphocytes mediates migration to the small intestine in response to a gradient of CCL25. CCL25-stimulated activation of α4β7 integrin promotes cell adherence to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expressed by vascular endothelial cells of the intestine, further mediating gut-specific homing. Inflammatory bowel disease is a chronic inflammatory condition that primarily affects the gastrointestinal tract and is characterized by leukocyte infiltration. Glucocorticoids (GCs) are widely used to treat inflammatory bowel disease but their effect on intestinal leukocyte homing is not well understood. We investigated the effect of GCs on the gut-specific chemokine receptor pair, CCR9 and CCL25. Using human peripheral blood-derived T lymphocytes enriched for CCR9 by cell sorting or culturing with all-trans retinoic acid, we measured chemotaxis, intracellular calcium flux, and α4β7-mediated cell adhesion to plate-bound MAdCAM-1. Dexamethasone (DEX), a specific GC receptor agonist, significantly reduced CCR9-mediated chemotaxis and adhesion to MAdCAM-1 without affecting CCR9 surface expression. In contrast, in the same cells, DEX increased CXCR4 surface expression and CXCL12-mediated signaling and downstream functions. The effects of DEX on human primary T cells were reversed by the GC receptor antagonist mifepristone. These results demonstrate that GCs suppress CCR9-mediated chemotaxis, intracellular calcium flux, and α4β7-mediated cell adhesion in vitro, and these effects could contribute to the efficacy of GCs in treating intestinal inflammation in vivo.

Unique pattern of PMN-MDSC migration in cancer

Abstract Myeloid-derived suppressor cells (MDSC) are pathologically activated immature myeloid cells that contribute to an immunosuppressive tumor microenvironment. In order to exert their effects at either the primary tumor site or in metastases, PMN-like MDSC (PMN-MDSC) must first migrate to these sites. There is some evidence to suggest that chemokine gradients can mediate their recruitment. We have observed that human PMN-MDSC accumulation in colon and lung tumors strongly correlates with the expression of S100A9, CXCL1 and CXCL8 in those tumors. Moreover, we have found that, in comparison to PMN, patient PMN-MDSC have an increased chemotactic response to CXCL8 in spite of decreased cell surface CXCR2 expression. Similarly, in murine transgenic models of melanoma, pancreatic and prostate cancer, we have found that PMN-MDSC migrate more than PMN when stimulated with CXCL1, in spite of similar levels of cell surface CXCR2 expression. Interestingly, murine PMN-MDSC are more migratory even without stimulation, and have greater speed, mean squared displacement and random motility coefficient than PMN. It is possible that in addition to specific factors secreted in the primary and metastatic tumor sites, PMN-MDSC recruitment to these sites is regulated by an altered migratory behavior of the cells themselves.

MP84-16 PHOSPHATIDYLINOSITOL 4-KINASE TYPE III? (PI4KA) AND CXCR4 CROSSTALK IN PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2016MP84-16 PHOSPHATIDYLINOSITOL 4-KINASE TYPE III? (PI4KA) AND CXCR4 CROSSTALK IN PROSTATE CANCER CELLS Michael Cher, Diego Sbrissa, Louie Semaan, Yanfeng Li, Assia Shisheva, and Srinivasa Chinni Michael CherMichael Cher More articles by this author , Diego SbrissaDiego Sbrissa More articles by this author , Louie SemaanLouie Semaan More articles by this author , Yanfeng LiYanfeng Li More articles by this author , Assia ShishevaAssia Shisheva More articles by this author , and Srinivasa ChinniSrinivasa Chinni More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2240AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The CXCR4 receptor directs homing of hematopoietic stem cells to CXCL12 ligand-rich bone marrow. Previous studies demonstrated that the CXCL12/CXCR4 axis promotes prostate cancer (PC) invasion and bone metastasis. We showed that CXCR4 expression is transcriptionally upregulated by TMPRSS2-ERG gene fusions in PC tumors, and its localization in lipid raft membrane microdomains promotes expression of invasive proteases. To identify novel lipid raft-associated CXCR4 regulators supporting invasion and metastasis, we performed proteomics analysis of lipid raft microdomains from stable CXCR4-overexpressing (CXCR4) and CXCR4-knockdown (shCXCR4) PC3 cell lines generated by lentiviral infection. METHODS We used stable isotope labeling by amino acids in cell culture (SILAC) proteomics, sucrose gradient buoyant density ultracentrifugation, fluorescence activated cell sorting (FACS), knockdown by siRNA silencing, cell invasion/growth assays, [2-3H]inositol cell labeling, SDS PAGE, Western blotting (WB), and inositol-lipid HPLC profiling. RESULTS SILAC identified phosphatidylinositol 4-kinase IIIα (PI4KA) to be highly associated with CXCR4 cells, along with slightly elevated SAC1 phosphatase. WB confirmed that expression of these phosphoinositide-regulating enzymes is associated with CXCR4 overexpression, thus anticipating their promotion of invasion and metastasis. Several lines of data demonstrated a link between PI4KA and CXCR4 signaling: PI4KA knockdown (i) reduced surface CXCR4 expression and CXCL12-stimulated invasion of CXCR4 cells; (ii) blocked CXCR4/AKT signaling lowering pAKT(Ser473) and pCXCR4(Ser339); and (iii) dropped global cellular PI4P levels. Unexpectedly, PI4KA knockdown resulted in a rise in PI4,5P2, suggesting a homeostatic response. Conversely, CXCR4 knockdown showed an expected increase in PI4,5P2 but an unexpected decrease in PI4P. Importantly, human PC tumor microarray data showed higher PI4KA expression in metastatic vs. localized PC or normal adjacent tissue. CONCLUSIONS These data confirm that PI4KA upregulates CXCR4 expression by altering inositol lipid levels thereby promoting CXCR4/AKT signaling, and that inhibition of CXCR4 signaling via PI4KA knockdown can suppress PC growth/invasion. Thus, PI4KA inhibitors may beneficial in patients with prostate cancer. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1095 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Michael Cher More articles by this author Diego Sbrissa More articles by this author Louie Semaan More articles by this author Yanfeng Li More articles by this author Assia Shisheva More articles by this author Srinivasa Chinni More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Isolation of an HIV-1 neutralizing peptide mimicking the CXCR4 and CCR5 surface from the heavy-chain complementary determining region 3 repertoire of a viremic controller.

The recent identification of neutralizing antibodies able to prevent viral rebound reemphasized the interest in humoral immune responses to control HIV-1 infection. In this study, we characterized HIV-1-inhibiting sequences from heavy-chain complementary determining region 3 (HCDR3) repertoires of a viremic controller. IgM and IgG-derived HCDR3 repertoires of a viremic controller presenting plasma-neutralizing activity and characterized by over 20 years of infection with a stable CD4 T-cell count were displayed on filamentous phage to identify HCDR3 repertoire-derived peptides inhibiting HIV-1 entry. Screening of phage libraries against recombinant gp120 led to the identification of an HCDR3-derived peptide sequence (LRTV-1) displaying antiviral properties against both X4 and R5 viruses. The interaction of LRTV-1 with gp120 was enhanced upon CD4 binding and sequence comparison revealed homology between LRTV-1 and the second extracellular loop of C-X-C chemokine receptor type 4 (CXCR4) (11/23) and the N-terminus of C-C chemokine receptor type 5 (CCR5) (7/23). Alanine scanning experiments identified different clusters of residues critical for interaction with the viral envelope protein. LRTV-1 peptide is to date the smallest human HCDR3 repertoire-derived peptide identified by phage display inhibiting HIV entry of R5 and X4 viruses. This peptide recognizes a CD4-dependent gp120 epitope critical for coreceptor binding and mimics the surface of CXCR4 and CCR5. Our data emphasize the potential of human HCDR3 immune repertoires as sources of small biologically active peptides for HIV cure.

Evaluation of assay interference and interpretation of CXCR4 receptor occupancy results in a preclinical study with MEDI3185, a fully human antibody to CXCR4.

BackgroundReceptor occupancy (RO) assays provide a means to measure the direct interaction of therapeutics with their cell surface targets. Free receptor assays quantify cell‐surface receptors not bound by a therapeutic while total receptor assays quantify the amount of target on the cell surface.MethodsWe developed both a flow cytometry‐based free RO assay to detect free surface CXCR4, and a total surface CXCR4 assay. In an effort to evaluate potential displacement interference, we performed in vitro experiments to compare on‐cell affinity with the IC50 values from in vitro and in vivo from the free CXCR4 assay. We determined free and total surface CXCR4 on circulating blood cells in cynomolgus monkeys dosed with MEDI3185, a fully human monoclonal antibody to CXCR4.ResultsWe devised an approach to evaluate displacement interference during assay development and showed that our free assay demonstrated little to no displacement interference. After dosing cynomolgus monkeys with MEDI3185, we observed dose‐dependence in the magnitude and duration of receptor occupancy and found CXCR4 to increase on lymphocytes, monocytes, and granulocytes. In a multiple dose study, we observed time points where surface CXCR4 appeared fully occupied but MEDI3185 was not detectable in serum. These paradoxical results represented a type of assay interference, and by comparing pharmacokinetic, ADA and total CXCR4 results, the most likely reason for the free CXCR4 results was the emergence of neutralizing anti‐drug antibodies (ADA). The total CXCR4 assay was unaffected by ADA and provided a reliable marker of target modulation in both in vivo studies. © 2015 The Authors Cytometry Part B: Clinical Cytometry Published byWiley Periodicals, Inc.

BRD4 Proteolysis Targeting Chimera (PROTAC) Leads to Sustained Degradation of BRD4 with Broad Activity Against Acute Leukemias and Overcomes Stroma Mediated Resistance By Modulating Surface Expression of CXCR4

Background: Hematological neoplasms are characterized by aberrant epigenetic events that modify the chromatin regulatory machinery to enhance oncogene expression (Zuber et al. Nature 2011). BRD4, a member of the bromodomain and extra terminal domain (BET) family, is critical in the assembly of a 'super enhancer complex' that drives expression of oncogenes MYC, CCND1, SOX2 and NF-kB and anti-apoptotic proteins e.g. Bcl-2 and BCL-XL (Klapproth et al. Br J Haematol 2010; Wang et al. Cancer Res 2014; Zou et al. Oncogene 2014). Small molecule BRD4 inhibitors lead to rapid accumulation of BRD4 that may partially account for their moderate suppression of MYC (Lu et al. Chem Biol 2015).ARV-825 is hetero-bifunctional PROTAC (Proteolysis Targeting Chimera) that recruits BRD4 to the E3 ubiquitin ligase cereblon, leading to fast, efficient, and prolonged degradation of BRD4 and sustained down-regulation of MYC (Lu et al. Chem Biol 2015).Aim: We investigated the activity of ARV-825 against leukemia cell lines representing acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) including gamma secretase inhibitor (GSI) resistant T-ALL and against primary AML blasts and compared it to that of the BRD4 inhibitor, JQ1. As stroma renders leukemia resistant, we tested if and through what mechanism ARV-825 can overcome stroma mediated drug resistance.Results: The IC50s for all tested cell lines at 72 hours were in very low nanomolar range; AML-OCIAML3-8.3 nM, HL60-2.4nM; ALL-KOPT-K1/SUPTI-1.3nM (GSI resistant T-ALL), LOUCY-2.3nM (early T-cell phenotype ALL), MOLT4-3.12nM and HPB-ALL-4.6nM (T- ALL) while the same for JQ1 was 102.6 nM, 5 nM, 1.5nM/8.8 nM, 4.5nM, 9.4 and 8.4 nM respectively. Consistent with sustained degradation of BRD4, the apoptotic effect of ARV-825 is sustained longer than that of JQ1 after the removal of compounds following a 24-hr treatment at IC50 (Annexin V positive 70% vs. 4% at 48 hrs post-washout respectively) (Fig. 1). Importantly, IC50 values for primary AML samples for ARV-825 were approximately 6-100 times lower than those for JQ1 (Fig. 2).ARV-825 led to sustained decreases of BRD4 and MYC proteins with corresponding increase in cleaved PARP, cleaved Caspase-3 and γH2AX in MOLT4 and OCIAML3 cells. While Bcl-2 level remained suppressed with ARV-825, it recovered at 48 hrs of treatment with JQ1.We co-cultured OCI-AML3 cells with normal bone marrow derived mesenchymal stromal cells (MSCs) to mimic the bone marrow microenvironment. While MSC protected OCI-AML3 cells from cytarabine (25 % reduction in cell death), there was no stromal protection for ARV-825. To gain mechanistic insight, we used a Mass cytometry (CyTOF) based approach to analyze the changes in apoptotic, cell adhesion and signaling proteins (panel of 24 antibodies) in OCI-AML3 cells after 12 and 24-hour treatment with ARV-825. MYC and surface CXCR4 were the two most down-regulated proteins. The reduction of surface CXCR4 was confirmed by conventional flow cytometry (Fig. 3A) and the functional relevance was confirmed in migration assays against the CXCR4 ligand SDF-1 (29.2% Control vs. 9.03% ARV-825) (Fig. 3B).Conclusion: ARV-825, a BRD4 PROTAC, has substantial anti-leukemia activity across a range of acute leukemias and importantly can overcome stroma-mediated drug resistance. ARV-825 promises to be an exciting molecule in the quest towards efficient BRD4 inhibition. [Display omitted] DisclosuresKonopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Lu:Arvinas, LLC: Employment, Equity Ownership. Qian:Arvinas, LLC: Employment, Equity Ownership.

The Peptidic CXCR4 Antagonist, BL-8040, Significantly Reduces Bone Marrow Immature Leukemia Progenitors By Inducing Differentiation, Apoptosis and Mobilization: Results of the Dose Escalation Clinical Trial in Acute Myeloid Leukemia

Background: The bone marrow (BM) niche protects acute myeloid leukemia (AML) cells from chemotherapy. BM homing of AML cells is dependent on CXCR4 and its ligand CXCL12; high levels of CXCR4 expression correlate with poor survival in AML. It is postulated that blocking the CXCL12/CXCR4 axis with a potent CXCR4 antagonist will disrupt the interaction of the leukemic blasts with the BM and augment the anti-leukemic effect of chemotherapy. BL-8040 (BKT140) is a short synthetic peptide which inhibits the binding of CXCR4 to CXCL12, resulting in the mobilization of leukemic blasts from the bone marrow to the peripheral blood. BL-8040 also inhibits CXCR4/CXCL12 mediated pro-survival ERK/Akt signaling and alters balance of pro/anti-apoptotic Bcl-2 proteins. BL-8040 has strong affinity for the CXCR4 receptor with long receptor occupancy, and, in contrast to other CXCR4 inhibitors, as a single agent induces apoptosis of leukemia cells ex-vivo in clinical samples. A clinical trial assessing the safety and efficacy of BL-8040 in combination with cytarabine (Ara-C) for the treatment of adult relapsed/refractory AML patients is currently ongoing (NCT01838395).Objective: To assess the safety, efficacy and pharmacodynamics/pharmacokinetic parameters of BL-8040 in combination with Ara-C in first or second relapse and refractory AML patients.Method: The study includes a dose escalation phase (3+3 design) followed by an expansion phase. Each patient receives a once daily SC dose of BL-8040 monotherapy on days 1-2 followed by the same dose of BL-8040 plus Ara-C (1.5 g/m2 for patients ≥60; 3 g/m2 for patients <60) on days 3-7. Five dose levels of BL-8040 (0.5, 0.75, 1, 1.25 and 1.5 mg/kg) have been tested. Extensive pharmacodynamic parameters such as CXCR4 expression on the leukemic cells, receptor occupancy, mobilization of leukemic cells and induction of apoptosis are being assessed after monotherapy with BL-8040 using peripheral blood sampling (PB) and BM aspirates at baseline and on day 3 prior to Ara-C administration. Clinical response to treatment is evaluated by BM biopsy on day 30 (-/+ 2).Results: Twenty two patients (median age, 61 y; range, 43-74 y) with relapsed/refractory AML were treated during the escalation phase. BL-8040 was escalated up to 1.5 mg/kg (recommended phase 2 dose) without reaching the MTD. Combination of BL-8040 with Ara-C was safe and well tolerated at all doses tested. Only one SAE (Sweet Syndrome) was reported as possibly related to study drug. The primary treatment related AEs were transient injection site and systemic reactions. The composite complete remission including complete remission (CR) and complete remission with incomplete blood count recovery (CRi) rate was 38% in subjects receiving BL-8040 doses of 1 mg/kg and higher (n=16). Two days of BL-8040 monotherapy triggered an average 40.2-fold (range 1.0-350.9) mobilization of immature AML progenitors (CD45+Low/CD34+/CD117+/HLA-DR+) from the BM to the PB. Moreover, a significant apoptotic effect on the immature leukemia progenitors in the BM following 2 days of BL-8040 monotherapy was noted. Flow cytometric evidence of apoptosis by BL-8040 was corroborated by an increase in cleaved caspase 3 positive blasts in day 3 bone marrow biopsies. In addition, BL-8040 treatment resulted in a median decrease of 57.7% (range, -10.4% to -96.2%) in the number of BM Leukemia progenitor cells (CD45+Low/CD34+/CD117+/HLA-DR+ out of total CD45+/CD34+ normal/progenitor cells) compared with the baseline biopsy. This decrease was accompanied with a 3.1 fold increase in granulocytes in the day 3 BM biopsy supporting differentiation effect. Finally, long receptor occupancy (up to 24 hours post dosing) was confirmed by FACS analysis measuring surface CXCR4 staining. Pharmacokinetics analysis confirmed dose incremental BL-8040 exposure with a short plasma half-life.Conclusions: The current data demonstrate that sustained blockade of the CXCR4-CXCL12 axis with BL-8040 has potent anti leukemic activity and in combination with Ara-C may improve the response typically achieved in this advanced AML population. Mechanistically, BL-8040 was able to induce apoptosis of leukemic progenitor cells, overcoming the stroma protection, inducing mobilization and differentiation. DisclosuresRowe:BioLineRx Ltd.: Consultancy; Amgen: Consultancy; BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Uy:Novartis: Research Funding. Altman:Seattle Genetics: Consultancy; Novartis: Consultancy; Spectrum: Consultancy; Astellas: Consultancy; Ariad: Consultancy; BMS: Consultancy. Peled:Biokine Ltd.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; BiolineRX: Consultancy, Research Funding. Pereg:BioLineRx: Employment. Vainstein:BioLineRx: Employment. Aharon:BioLineRx: Employment. Pemmaraju:Stemline: Research Funding; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria. Cortes:Teva: Research Funding; Pfizer: Consultancy, Research Funding; BMS: Consultancy, Research Funding; BerGenBio AS: Research Funding; Novartis: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy.

CXCR4 Gene Dosage Is Critical for HSC Engraftment

Introduction: Warts, Hypogammaglobulinemia, Infections and Myelokathexis Syndrome (WHIMS) is an autosomal dominant immunodeficiency resulting from gain-of-function mutations in the chemokine receptor CXCR4. We recently described a unique WHIMS patient who underwent spontaneous genetic and phenotypic reversion at approximately age 30 after being severely affected as a child. Her reversion was due to a single catastrophic genetic event known as chromothripsis (chromosome shattering) resulting in the deletion of one copy of 163 genes in addition to her mutant copy of CXCR4 on chromosome 2. This event was traced to a hematopoietic stem cell (HSC) that had spontaneously repopulated her bone marrow; however, which of the genes was responsible and the mechanism required further investigation.Methods: Mouse models of CXCR4 haploinsufficiency (Cxcr4+/o) and WHIMS (Cxcr4+/S338X) were used in competitive bone marrow repopulation experiments transplanting whole bone marrow cells or purified HSC. Recipient mice were treated with / without lethal irradiation prior to transplant. Genome editing with TALENs and CRISPR-Cas9 technology was used to target CXCR4 for deletion in human cell lines.Results:Cxcr4 haploinsufficiency markedly enhanced HSC engraftment potential in recipient WHIM mice whether the donor HSC were purified from whole bone marrow cells or not, and whether the recipient was conditioned by lethal irradiation or not. Enhanced engraftment by Cxcr4 haploinsufficient donor HSC also occurred in wild-type mouse recipients, but to a lesser extent, and was also HSC intrinsic. Genome editing experiments have been successful at deleting one or both copies of CXCR4 in human cell lines in up to 40% of treated cells, and in reducing CXCR4 surface expression.Conclusion: While CXCR4 was already understood to be important in HSC biology, this patient and subsequent murine experiments have proven that the gene dosage of CXCR4 is a critical factor affecting HSC engraftment. Genome editing is a promising technology for deleting one copy of CXCR4, ideally the WHIM allele,in autologous HSC as a strategy to cure WHIM syndrome. DisclosuresMcDermott:US National Institutes of Health: Employment, Patents & Royalties: pending. Jacobs:US National Institutes of Health: Employment, Patents & Royalties: pending. Liu:US National Institutes of Health: Employment, Patents & Royalties: pending. Gao:US National Institutes of Health: Employment, Patents & Royalties: pending. Murphy:US National Institutes of Health: Employment, Patents & Royalties: pending.

The Dual PIM/PI3-K Inhibitor Ibl-202 Overcomes Microenvironmental Mediated Resistance in Multiple Myeloma and Prevents PIM1 Induced CXCR4 Upregulation

Background:The interaction of multiple myeloma (MM) cells with bone marrow (BM) cells along with factors in the BM milieu such as chemokines and cytokines play a crucial role in both progression of MM and drug resistance. Activation of the PI3-K/Akt survival pathway is a characteristic of both human MM cell lines and patient samples. This activation can be linked to BM microenvironmental signalling and use of proteasome inhibitors in treatment, suggesting this as a crucial point of therapeutic intervention to abrogate growth and survival signals in MM. However, the efficacy of such therapeutics has been modest and is likely to be compromised by the stimulation of compensatory signalling pathways, such as the PIM kinases, which like the PI3-K/Akt pathway are also induced by BM microenvironmental influences and share similar downstream targets. These proto-oncogenic kinases are constitutively active and play an important role in proliferation and survival in MM. The influence of these kinases on homing and migration has been observed in other malignancies, this has yet to be reported in MM. Here we report the effects of a dual inhibitor of PIM/PI3-K, IBL-202, and provide novel insights into effects on cell survival, signaling and migration.Methods:We investigated the effect of IBL-202 against a panel of MM cell lines (MM.IS, NCI-H929s, KMS11 and RPMI-8226) and primary MM patient samples. The in vitro efficacy of IBL-202 was compared to that of single pan-PIM inhibitors pPIMi and AZD1208 and also the pan-PI3-K inhibitor GDC-0941. Apoptosis was measured with AnnexinV staining and cell cycle analysed with Edu/DAPI staining.To mimic BM microenvironmental conditions MM cells were cultured under hypoxic conditions (1% O2) and in co-culture with the human stromal cell line HS5.Surface expression of CXCR4 was assessed in MM cell lines by flow cytometry. PIM kinases, pCXCR4 and downstream targets of PIM/PI3-K were examined by western blot. Transwell migration assays were carried out in the presence of 50ng SDF-1α for 4h @ 37o C.Results:Simultaneous inhibition of PIM and PI3-K using IBL-202 in vitro was significantly more potent at inducing apoptosis than GDC-0941, pPIMi or AZD1208 in all MM cell lines tested. IC50 values were under 1μM for IBL-202 at 48h whilst in comparison the pan PIM inhibitors pPIMi and AZD1208 scored IC50 values between 5 and 10μM. The IC50 for GDC-0941 was on average 5μM (Figure 1).At the molecular level there was a notable decrease in phosphorylation of known PIM/PI3-K targets Akt (Ser473), Bad (Ser112) and the translational targets S6 (Ser235/236) and 4EBP1 (Thr37/46). The levels of total proteins were unchanged.Treatment with increasing doses of IBL-202 led to a marked reduction in cells in S phase of the cell cycle. These changes were paralled by down regulation of the cell cycle promoting proteins cyclin D1 and c-myc.IBL-202 was also effective in inducing apoptosis in primary MM patient samples (n=4) after just 24h as assessed by Annexin-V staining (Figure 2).To explore the role of the BM microenvironment we co-cultured MM cell lines with HS5s. This led to strong induction of PIM2 in MM cells. While MM cells in this setting were protected from Bortezomib-induced cell death, the apoptotic effect of IBL-202 was enhanced. In a further effort to mimic the tumour microenvironment we cultured MM cell lines in hypoxia. This may be of particular relevance as Pim-1 has been reported to be a pivotal regulator involved in hypoxia-induced chemoresistance. MM cells were further sensitised to IBL-202 in hypoxia. In addition, hypoxia increased the surface expression of CXCR4, a chemokine receptor critical for homing of MM cells to the bone marrow, with a concomitant increase in PIM1. Treatment of MM cell lines with IBL-202 reduced the level of PIM1 and CXCR4 Ser339 phosphorylation, along with down regulation of CXCR4 surface expression resulting in reduced migration of MM cells along an SDF-1 gradient.Conclusion:Together these data provide direct evidence of the potency of IBL-202 in MM in conditions that mimic the BM microenvironment. Moreover, they indicate a potential role for PIM kinases in facilitating dissemination and invasiveness of MM by CXCR4 and provide an added rationale for targeting PIM kinases in MM. [Display omitted] [Display omitted] DisclosuresO'Neill:Inflection Biosciences: Employment.

Identification of CXCR4 Expression By Targeted PET/MR Imaging in Patients with Acute Myeloid Leukemia (AML)

IntroductionAML is an aggressive hematological neoplasm originating from hematopoietic precursor or stem cells and is rapidly fatal if untreated. Although rates of complete remission (CR) approach 80% in patients with favorable risk profile according to European Leukemia Net classification, CR rates drop dramatically within the intermediate or adverse prognostic groups, and most patients relapse in spite of CR. The origin of refractory or relapsed disease is - according to several studies - the bone marrow (BM) niche that protects the leukemia initiating cells (LIC) from cytotoxic drugs.The chemokine receptor CXCR4 is a key factor mediating the crosstalk between the BM niche and LICs. CXCR4 is a G-protein coupled chemokine receptor with Stromal cell-derived factor-1α (SDF-1α) as its single known chemokine-ligand, and high expression is associated with poor prognosis in AML. Targeting the CXCR4/SDF-1αaxis with small peptide inhibitors or monoclonal antibodies has shown promising results in preclinical and clinical studies, thus rendering it an attractive target for future therapeutic strategies (Peled & Tavor, Theranostics, 2014). In this observational study we analyzed whether the new CXCR4-targeted positron emission tomography (PET)-probe [68Ga]-Pentixafor (Philipp-Abbrederis et al, EMBO Mol Med, 2015; Wester et al, Theranostics, 2015) is applicable for molecular imaging of patients with AML with the goal to develop a CXCR4- targeted peptide-receptor-radiotherapy (PRRT).MethodsUsing myeloid malignancies (MDS, active AML) as an exemplary CXCR4-expressing cancer entity we imaged 10 patients with the CXCR4 specific PET-probe [68Ga]-Pentixafor by whole body PET/magnetic resonance imaging (PET/MR) after they signed informed consent for this observational assessment. Maximum standardized uptake values (SUVmax) of the involved BM areas were compared to BM from patients without BM malignancy assessed in a different study. CXCR4 surface expression of leukemic BM cells of an unselected cohort of 67 patients with myeloid malignancies was analyzed by flow cytometry. CXCR4 expression in 14 established AML cell lines was measured by flow cytometry and quantitative PCR.Results Out of 10 patients who were imaged with [68Ga]-Pentixafor-PET/MR, 4 - all of them with the diagnosis of an AML - showed visually positive PET signal of the BM. Those areas correlated well with disease infiltration as determined by MR imaging and the meanSUVmax was significantly higher as compared to visually CXCR4 negative patients (meanSUVmax 8.23 ± 5.23 vs. 2.26 ± 0.47; p=0.036). One of the 10 patients presenting with proven extramedullary relapse showed a CXCR4 positive PET signal within the lesion (SUVmax 5.12), whereas the cytological non-involved BM was PET negative (meanSUVmax 1.82). CXCR4 surface expression in established AML cell lines was variable and ranged from low to high, and correlated with mRNA levels. These data reflect the different expression values we found in the cohort of patients with myeloid malignancies. There was a trend to higher CXCR4 surface expression on blasts when AML patients were compared to MDS patients (median fluorescent intensity CXCR4/isotype 1.73 ± 0.25 vs. 0.96 ± 0.06; p=0.063) that became significant when blast frequency exceeds 30% (≤30% vs. >30% blasts: 1.07 ± 0.11 vs. 2.08 ± 0.38; p=0.006). In this patient cohort we did not observe any correlation between CXCR4 expression and factors such as diagnosis (secondary vs. de novo AML), disease status (refractory or relapse vs. no pretreatment), molecular subtypes or cytogenetic aberrations.DiscussionImaging of CXCR4 by PET/MR with [68Ga]-Pentixafor is feasible in patients with AML. Due to the higher uptake in leukemic BM compared to control BM and its low uptake in other organs (e.g. liver, brain and gut), CXCR4 could serve as an attractive new PRRT-target in selected patients with AML. Such PRRT approaches are of special interest in the setting of allogeneic BM transplantation as conventional myeloablative conditioning regimens - although they are associated with a better relapse free survival - show substantial rates of transplant-related mortality and are therefore not applicable in many AML patients due to their age. In particular, therapeutic targeting of CXCR4 by PRRT provides a unique opportunity of integrating a LIC/niche-directed treatment into the conditioning regimen. Such clinical studies are in development. DisclosuresWester:Scintomics GmbH: Other: General Manager. Keller:Roche: Consultancy, Honoraria; Pfizer: Consultancy.