CXCL12-CXCR4 Axis Research Articles

Abstract B13: Hypoxia-induced chemokine activation and lymphnode metastases in primary cervix xenografts

Abstract Aim: To examine the role of the chemokine pathway in cervix cancer in the regulation of hypoxia-induced metastases and bone marrow derived myeloid cells (BMDCs) in the tumor microenvironment. Methods: Cervix cancer biopsies from patients in clinical studies at PMH were used for implantation into the cervix of NRG mice. Relative CXCR4 expression by qRT-PCR in biopsies from 117 patients with cervical cancer and 17 primary, orthotopic, cervical cancer xenografts was conducted. Mice bearing orthotopic xenografts were exposed to cyclic hypoxia (12x 10min cycles of breathing 7%O2 or air; 4hr/day for 2 wks) during their growth in combination with AMD3100 (Plerixafor), administered by osmotic pumps (5mg/kg/day) implanted subcutaneously for 3 weeks. Primary tumor size and para-aortic lymph node metastases were assessedfor metastases (H&E staining). Mice received 15x30Gy (2Gy daily 5x/wk) targeted radiotherapy with cisplatin (4mg/kg/wk)(RTCTx) with or without AMD3100. The mice were monitored weekly for tumor growth by CT scanning. Immunohistochemistry was used to analyse levels of hypoxia (EF5/CA-9), blood (CD31) and lymphatic (LYVE-1) vessels, and CXCR4/CXCL12 in the orthotopic tumors and involved lymphnodes. Cd11b staining was used to assess neutrophil infiltration into the tumors. The GI toxicity of AMD3100 with RTCTx was assessed by gut crypt assay. Results: The (early passage) primary orthotopic cervical xenografts used in this study invade and metastasize to lymph nodes in a manner similar to cervical cancer in patients. The microenvironmental features (vascularity, stroma content, hypoxia, IFP) of the xenografts match the original biopsy. The variability in baseline CXCR4 gene expression (by qRT-PCR) in our xenograft models is also similar to that seen in the patient biopsies. Preliminary data also shows comparable CXCL12 and CD11b+ BMDC infiltration in a patient biopsy and in primary xenografts. Induction of tumor hypoxia by exposing the animals to a low oxygen environment increased CXCL12 and CXCR4 gene and protein expression and resulted in a significant increase in the number of lymph node metastases. Treatment with the CXCR4 antagonist AMD3100 reversed these effects. Tumor-bearing animals treated with targeted RTCTx combined with AMD3100 demonstrated significantly enhanced growth delay compared to RTCTx alone and demonstrated reduced metastases at the end of the study. There was no evidence that AMD3100 enhanced the gut toxicity of the RTCTx treatment. Conclusions: Our results suggest that CXCR4 plays an important role in metastasis and tumor growth, under hypoxic conditions, in human cervical cancer. Targeting the CXCR4-CXCL12 axis, with chemokine specific inhibitors,may serve as a novel and attractive therapeutic approach in cervix cancer.These studies provide a strong focus on developing and optimizing new targeted therapeutic approaches aimed at mitigating the adverse consequences of altered chemokine/BMDC signaling and translating these to high-impact phase I/II clinical trials. Citation Format: Naz Chaudary, Salomeh Jelveh, Patricia Lindsay, Melania Pintilie, Michael Milosevic, Richard P. Hill. Hypoxia-induced chemokine activation and lymphnode metastases in primary cervix xenografts. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B13. doi:10.1158/1538-7445.CHTME14-B13

A biologically inspired lung-on-a-chip device for the study of protein-induced lung inflammation.

This study reports a biomimetic microsystem that reconstitutes the lung microenvironment for monitoring the role of eosinophil cationic protein (ECP) in lung inflammation. ECP induces the airway epithelial cell expression of CXCL-12, which in turn stimulates the migration of fibrocytes towards the epithelium. This two-layered microfluidic system provides a feasible platform for perfusion culture, and was used in this study to reveal that the CXCL12-CXCR4 axis mediates ECP induced fibrocyte extravasation in lung inflammation. This 'lung-on-a-chip' microdevice serves as a dynamic transwell system by introducing a flow that can reconstitute the blood vessel-tissue interface for in vitro assays, enhancing pre-clinical studies. We made an attempt to develop a new microfluidic model which could not only simulate the transwell for studying cell migration, but could also study the migration in the presence of a flow mimicking the physiological conditions in the body. As blood vessels are the integral part of our body, this model gives an opportunity to study more realistic in vitro models of organs where the blood vessel i.e. flow based migration is involved.

Targeting the CXCR4 Pathway: Safety, Tolerability and Clinical Activity of Ulocuplumab (BMS-936564), an Anti-CXCR4 Antibody, in Relapsed/Refractory Acute Myeloid Leukemia

Background: CXCR4 is a chemokine receptor over-expressed on > 75% of cancers, including myeloid leukemia blasts. Ulocuplumab (BMS- 936564) is a first in class, fully human IgG4 monoclonal antibody which inhibits the binding of CXCR4 to CXCL12, resulting in the mobilization of leukocytes from the bone marrow to the peripheral blood. Ulocuplumab also induces apoptosis of CXCR4+ cells in clinicial samples (i.e., leukemia blasts) and in in vitro experiments (e.g. in Tregs). It is thus hypothesized that Ulocuplumab may induce the mobilization and apoptosis of myeloid blasts & immunosuppressive cells which could improve the overall response rate to chemotherapyObjective: To conduct a first-in-man phase I dose escalation/ expansion trial to determine the safety, pharmacology, and clinical benefit of Ulocuplumab in relapsed/refractory AMLResults: Seventy three subjects (median age, 58 yrs; range, 21-79 y) with relapsed/refractory AML were treated with Ulocuplumab and MEC [mitoxantrone, 8 mg/m2; etoposide , 100 mg/m2; and cytarabine, 1 g/ m2; i.v. d 1-5]. Thirty subjects in escalation received a single infusion of Ulocuplumab (doses of 0.3, 1, 3, or 10 mg/kg) one week prior to starting MEC and 3 additional weekly doses per MEC cycle thereafter (starting on Day 1). Ulocuplumab was escalated to a maximum of 10 mg/kg without any dose-limiting toxicity during monotherapy or in combination with MEC in the 1st cycle. In expansion phase, 43 patients were similarly treated with10 mg/kg Ulocuplumab and MEC. The overall complete remission and complete remission with incomplete blood count recovery rate (CR/CRi) was 51%. Subjects with first CR > 6 months had better ORR (16/23, 70%) than those with CR1 ≤6 months or primary induction failure (6/20, 30%). Of note, four subjects had CR/CRi after a single dose of Ulocuplumab monotherapy. Transient, mild/moderate thrombocytopenia was the only treatment-related AE documented with Ulocuplumab monotherapy. Only one subject presented a mild infusion reaction on Cycle 1 Day 1. The safety profile in combination with MEC was similar to MEC alone as was the 60 day all-cause mortality (16.3%). A median 2- and 5-fold mobilization of leukocytes and leukemic blasts into the peripheral circulation was reported at day 8, respectively. There was a trend demonstrating that higher CXCR4 expression on AML blasts correlated with a positive clinical response. Reversible and manageable hyperleukoctosis occurred in one subject.Conclusions: This study shows that the blockade of the CXCR4-CXCL12 axis with Ulocuplumab has antileukemic activity and safely improves the historic response rate achieved with MEC alone (i.e.,24-28%). DisclosuresBecker:Bristol-Myers Squibb: Research Funding. Off Label Use: Etoposide is indicated in the management of refractory testicular tumors and small cell lung cancer. . Foran:Bristol-Myers Squibb: Research Funding. Altman:Bristol-Myers Squibb: Research Funding. Yacoub:Bristol-Myers Squibb: Research Funding. Castro:Bristol-Myers Squibb: Research Funding. Sabbatini:Bristol-Myers Squibb: Employment. Dilea:Bristol-Myers Squibb: Employment. Wade:Bristol-Myers Squibb: Employment. Xing:Bristol-Myers Squibb: Employment. Gutierrez:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Smith:Bristol-Myers Squibb: Research Funding.

Phase Ib Study of the Novel Anti-CXCR4 Antibody Ulocuplumab (BMS-936564) in Combination with Lenalidomide Plus Low-Dose Dexamethasone, or with Bortezomib plus Dexamethasone in Subjects with Relapsed or Refractory Multiple Myeloma

Background: CXCR4 is a chemokine receptor over-expressed on > 75% of cancers, including malignant plasma cells. Ulocuplumab (BMS- 936564) is a first in class, fully human IgG4 monoclonal anti-CXCR4 antibody which inhibits the binding of CXCR4 to CXCL12. Ulocuplumab induces apoptosis of CXCR4+ multiple myeloma cell lines and has single agent activity in vivo in MM tumor xenograft models. It is thus hypothesized that Ulocuplumab may improve the overall response rate to standard therapy in relapsed-refractory multiple myeloma (rel/ref MM) by distinct mechanisms of action, i.e., mobilization and apoptosis of malignant plasma cells and immune regulation.Objective:This study aimed to determine the safety, tolerability, pharmacokinetics, pharmacodynamics and clinical activity of Ulocuplumab alone and in combination with lenalidomide plus low-dose dexamethasone (Len-Dex), or in combination with bortezomib plus dexamethasone (Bor-Dex) in subjects with rel/ref MM.Methods: Ulocuplumab (i.e., 1, 3 and 10 mg/kg) was dose escalated with a 3-plus-3 design with doses of Len-Dex orBor-Dex to identify MTD. For Cycle 1, Ulocuplumab was administered as monotherapy on Days 1 and 8. Starting on Day 15, Ulocuplumab was administered in combination with lenalidomide [25 mg/d/21 days of a 28 day cycle] plus low dose dexamethasone [40 mg/week] and monitored for incidence of DLT(s) within Cycle 1 (42 Days) of study treatment. For the Bor-Dex group, also starting on Day 15, Ulocuplumab was administered in combination with bortezomib (1.3 mg/m2Days 1, 4, 8, 11 of a 21 day cycle] plus dexamethasone [days 1,2,4,5,8,9,11,12] and monitored for incidence of DLT(s) within Cycle 1 (35 Days) of study treatment. For the expansion phase, subjects received 10mg/kg Ulocuplumab monotherapy on Days 1 and 8 followed by weekly doses in combination with Len-Dex (28-day cycles). Subjects were assessed at day 14 and after every cycle by IMWG criteria.Results: Forty four subjects were evaluated (median age, 59.5 yrs; range, 44-77). The median number of prior therapies was 4, (range, 1-9), with 76% of subjects having received ≥ 3. Subjects had received bortezomib in 93% of the cases, lenalidomide in 86% , thalidomide in 30%, carfilzomib in 20% and pomalidomide in 11%. Thirty subjects in escalation received Ulocuplumab alone and in combination with Len-Dex : One subject in the U-Bor-Dex group experienced a DLT in which there was delayed platelet recovery to ≤ Grade 1 or baseline which resulted in a delay of dosing of ≥ 21 days. Ulocuplumab was escalated to a maximum of 10 mg/kg without reaching MTD in monotherapy or in combination therapy. Twenty one subjects were treated in expansion phase. There were no grade 4 toxicities with Ulocuplumab monotherapy and Grade 3 toxicities with monotherapy included thrombocytopenia (6.5%), anemia (4.3%), respiratory infections (4.3%), femur fracture (4.3%), lymphopenia (2.2%), neutrophil count decreased (2.2%), platelet count decreased (2.2%) and cerebrovascular accident (2.2%). The safety profile of Ulocuplumab with Len-Dex or Bor-Dex was similar to either combination alone. Two subjects (4.5%) presented reversible G2 infusion reactions. The overall response rate (≥ PR) for all subjects in escalation and expansion was 50% (22/44), including 1 CR, 6 VGPR and 15 PR. The ORR by group was 55.1 % (16/29) and 40% (6/15) for U-Len-Dex and U-Bor-Dex, respectively. Furthermore, the ORR in expansion with 10 mg/kg U-Len-Dex was 57% (12/21) with 4 VGPRs and 8 PRs. Eight subjects in this expansion group had at least SD with a mean duration of 159 days (range, 46-437 days), resulting in 95% of subjects with clinical benefit. A median 2-fold mobilization of leukocytes into the peripheral circulation was reported after each infusion of Ulocuplumab at 3 and 10 mg/kg. Samples showed rapid mobilization of leukocytes at 2 hours post-Ulocuplumab with a partial decrease at 3-4 days post-administration without reaching baseline. Mobilization of plasma cells was also documented in some subjects.Conclusions: This study shows that the blockade of the CXCR4-CXCL12 axis by Ulocuplumab is safe and shows a high response rate of over 50% in the Len-dex arm of patients with relapsed/refractory myeloma who have been previously treated with lenalidomide and bortezomib. The distinct mechanisms of action of this antibody make it a new class of anti-myeloma drug that deserves further exploration in clinical trials. DisclosuresGhobrial:Onyx: Advisory board Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding; Millennium: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity’s Board of Directors or advisory committees. Off Label Use: Plerixafor is not FDA approved for relapsed myeloma. Anderson:Celgene: Consultancy; Sanofi-Aventis: Consultancy; Onyx: Consultancy; Acetylon: Scientific Founder, Scientific Founder Other; Oncoprep: Scientific Founder Other; Gilead Sciences: Consultancy. Sabbatini:Bristol-Myers Squibb: Employment. Dilea:Bristol-Myers Squibb: Employment. Cardarelli:BMS: Employment. Wade:Bristol-Myers Squibb: Employment. Xing:Bristol-Myers Squibb: Employment. Gutierrez:Bristol-Myers Squibb: Employment. Cohen:BMS: Employment.

Investigation of the CXCL12-CXCR4/CXCR7 Axis in Multiple Myeloma (MM): Adhesion Molecules As Novel Targets of the Potent Proteasome Inhibitor Carfilzomib

Introduction: The interaction of malignant plasma cells (PC) with the bone marrow (BM) microenvironment (BMM) is fundamental for multiple myeloma (MM) pathogenesis, with BMM playing a key role in disease development, progression and patients’ symptoms. The CXCL12/CXCR4 axis is pivotal in this cross-talk. By activating CXCR4, CXCL12 drives MM cells to the protective BM and facilitates environment-mediated drug resistance. Whether or not CXCR7, the second receptor for CXCL12, is expressed by malignant PC has not been determined yet. In AML and CLL, the phosphorylation status of CXCR4 proved important: phosphorylation at Ser339 by PIM-1 kinase promoted re-externalization of CXCR4 and its reactivation to CXCL12 stimulation (Grundler et al. JEM 2009; Decker et al. Mol Cancer Therapeutics 2014).Methods: Human MM cell lines (MMCLs) U266, L363, NCI-H929 and primary BM specimens (patient samples n=9, median BM-infiltration: 30%; range: 15-90%) were used. MMCLs and primary MM patient specimens were assessed for CXCR4, CXCR7 and CD138 surface (s) expression via flow cytometry. For CXCR4 analysis, the conformation independent antibody clone 4G10 was used for both flow and image cytometry experiments. The MMCLs were cultured alone or with the stroma cell line M2-10B4, and treated with the novel proteasome inhibitor carfilzomib (20, 50, 100nM), either alone or combined with the CXCR4 antagonist AMD3100 (50µM) or anti-CXCL12 “Spiegelmer” NOX-A12 (100nM). Cell viability was assessed by PI/Annexin staining. CXCR4 downstream pathways and PIM-1 kinase expression were analyzed by Western blot.Results: All MMCLs and primary MM cells expressed sCXCR7. sCXCR7 expression was represented as mean (% of positive live cells) +/- SD: U266 (89.1 +/-7.1), L363 (82.1 +/-7.5) and NCI-H929 (82.4 +/-8.2) were largely CXCR7 positive. The sCXCR7 expression of unsorted primary BM cells was 21.7 +/- 10.5 (n=9), being slightly higher than their sCXCR4 expression: 14.6 +/- 7.5, (n=7). When analyzing only the sCD138 positively stained population, the malignant PC showed an even higher level of marker expression: sCXCR7: 78.4 +/- 26.6 (n=8) and sCXCR4: 65.5 +/- 39.1 (n=7).Within our in vitro coculture model (Udi J,..Engelhardt. Br J Haematol 2013; Zlei,…Engelhardt. Exp Hematol 2007; Schüler,..Engelhardt. Expert Opin Biol Ther 2013), CXCL12 stimulation of U266 led to CXCR4 internalization, CXCR4 phosphorylation at Ser339 and actin polymerization. The latter was abrogated by AMD3100 and NOX-A12, proving the functionality of CXCL12-CXCR4 in our MM model. In U266, CXCL12 did not induce CXCR7 internalization. Carfilzomib was rigorously tested in 3 different conditions: it proved cytotoxic for U266 and L363 with pulse (t=1h), continuous treatment (t=3d) and to a lesser extent in coculture (t=3d). Neither AMD3100 nor NOX-A12 significantly reversed the stroma protection. Apart from its cytotoxic effects, carfilzomib (t=3d) reduced sCD138 in U266 or L363 in coculture. As a pulse treatment (t=1h) in U266 monoculture, carfilzomib reduced sCD138 and sCXCR4 without affecting sCXCR7. A 1h pulse of carfilzomib had no effect on pERK/ERK (n=4), nor did it change CXCR4 protein levels (n=2). However, pCXCR4 (Ser339) and its phosphorylating kinase PIM-1 were significantly reduced by carfilzomib in a dose dependent fashion (n=4).Conclusion: CXCR7 is widely expressed on malignant PC and its contribution to the CXCL12-CXCR4 axis in MM is worthy of future study. Carfilzomib shows strong cytotoxic activity in monoculture, which cannot be completely reversed by stroma protection. Carfilzomib appears to downregulate sCXCR4 by reduction of pCXCR4 via decreased PIM-1 protein levels. These data could explain its potency and why the CXCR4/CXCL12-inhibitors AMD3100 and NOX-A12 do not induce additive cytotoxicity. Moreover, and to the best of our knowledge unknown to date, these results suggest a link between CXCR4 and PIM-1 in MM. Therefore, analyzing the (off-) target effects of anti-myeloma substances on the MM BMM interaction seems to be a promising approach to discover optimal drug combinations and new therapeutic targets. DisclosuresNo relevant conflicts of interest to declare.

Role of platelet-derived growth factor/platelet-derived growth factor receptor axis in the trafficking of circulating fibrocytes in pulmonary fibrosis.

Circulating fibrocytes have been reported to migrate into the injured lungs, and contribute to fibrogenesis via CXCL12-CXCR4 axis. In contrast, we report that imatinib mesylate prevented bleomycin (BLM)-induced pulmonary fibrosis in mice by inhibiting platelet-derived growth factor receptor (PDGFR), even when it was administered only in the early phase. The goal of this study was to test the hypothesis that platelet-derived growth factor (PDGF) might directly contribute to the migration of fibrocytes to the injured lungs. PDGFR expression in fibrocytes was examined by flow cytometry and RT-PCR. The migration of fibrocytes was evaluated by using a chemotaxis assay for human fibrocytes isolated from peripheral blood. The numbers of fibrocytes triple-stained for CD45, collagen-1, and CXCR4 were also examined in lung digests of BLM-treated mice. PDGFR mRNA levels in fibrocytes isolated from patients with idiopathic pulmonary fibrosis were investigated by real-time PCR. Fibrocytes expressed both PDGFR-α and -β, and migrated in response to PDGFs. PDGFR inhibitors (imatinib, PDGFR-blocking antibodies) suppressed fibrocyte migration in vitro, and reduced the number of fibrocytes in the lungs of BLM-treated mice. PDGF-BB was a stronger chemoattractant than the other PDGFs in vitro, and anti-PDGFR-β-blocking antibody decreased the numbers of fibrocytes in the lungs compared with anti-PDGFR-α antibody in vivo. Marked expression of PDGFR-β was observed in fibrocytes from patients with idiopathic pulmonary fibrosis compared with healthy subjects. These results suggest that PDGF directly functions as a strong chemoattractant for fibrocytes. In particular, the PDGF-BB-PDGFR-β biological axis might play a critical role in fibrocyte migration into the fibrotic lungs.

Abstract 4775: PI3K-p110β is required for leukemic transformation and HSC depletion in the absence of Pten

Abstract The tumor suppressor PTEN, which antagonizes PI3K signaling, plays critical roles in cell cycle, cell migration and stem cell self-renewal. PTEN is frequently mutated or inactivated in T-cell acute lymphoblastic leukemia (T-ALL) and acute myelogenous leukemia (AML). In mice, HSC-specific deletion of Pten leads to myeloproliferative phenotype, transplantable leukemia, and increased cycling of hematopoietic stem cells (HSCs), leading to their depletion. Rapamycin, an mTOR inhibitor, rescues this phenotype. Thus, it is likely that PI3K itself plays an important role in Pten-deficient HSCs and leukemic cells, but the specific contributions of the PI3K isoforms are poorly understood. We demonstrate that genetic ablation or pharmacological inhibition of PI3K-p110β prevents myeloid neoplasia and prolongs survival. Importantly, p110β ablation rescues HSCs in the bone marrow (BM), and reinstates long-term hematopoietic reconstitution. Moreover, Pten deficiency leads to aberrant expression of Cxcl12 in the BM and spleen, enabling the generation of an alternative splenic niche. Inactivation of p110β rescues retention of HSCs, and perturbs the splenic niche by interfering with the Cxcl12-CXCR4 axis. This suggests that selective inhibition of p110β offers an alternative therapeutic strategy in PTEN deficient leukemias while restoring untransformed HSCs to their proper niche. Citation Format: Haluk Yuzugullu, Lukas Baitsch, Allison Steiner, Linda K. Clayton, Kira Gritsman, Jean J. Zhao. PI3K-p110β is required for leukemic transformation and HSC depletion in the absence of Pten. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4775. doi:10.1158/1538-7445.AM2014-4775

Abstract LB-168: Perivascular, CXCR4-expressing macrophages in tumors promote relapse following chemotherapy

Abstract The relapse of cancer after frontline therapies like chemotherapy is a major clinical problem as tumors often grow back at the site of surgical removal and/or as metastases. Tumor-associated macrophages (TAMs) are a major cellular component of both primary and metastatic tumors, and promote such key processes in tumor progression as angiogenesis, immunosuppression, invasion, and metastasis. Gene expression profiling and immunostaining studies have identified both classically (M1) and alternatively (M2) activated TAMs in human and mouse tumors. Heavily M2-skewed TAMs - those expressing the angiopoietin receptor, TIE2 - have been shown to be important for angiogenesis and immunosuppression in mouse tumors. Moreover, high levels of TAMs expressing another M2 marker, CD163, correlate with poor prognosis in various forms of human cancer. TAMs have been shown recently to reduce the sensitivity of mouse mammary tumors to cytotoxic agents like paclitaxel (PTX) and doxorubicin. Here, we report on our use of various mouse tumor models to investigate the role of TAMs in tumor relapse after chemotherapy. Our studies show that a distinct subset of M2-skewed, CXCR4hi VEGF+ TAMs cluster around blood vessels after administration of various cytotoxic agents. This correlated with increased tumor CXCL12 levels - and the selective depletion of this perivascular TAM subset, using the CXCR4 antagonist, AMD3100 (Plerixafor), revealed an important role for these cells in driving revascularisation and regrowth in post-chemotherapy tumors. Additionally, adoptive transfer of this TAM subset into tumor-bearing mice after chemotherapy accelerated tumor regrowth. No such effect was seen with non-M2 skewed TAMs isolated from the same tumors. These studies suggest that combining chemotherapy with inhibitors of the CXCL12-CXCR4 axis could delay tumor relapse in cancer patients. Citation Format: Claire E. Lewis. Perivascular, CXCR4-expressing macrophages in tumors promote relapse following chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-168. doi:10.1158/1538-7445.AM2014-LB-168

Expression dynamics of CXCL12 and CXCR4 during the progression of mycosis fungoides.

Mycosis fungoides (MF) classically presents from patch stage to plaque stage over a number of years and finally progresses to tumour stage with nodal or visceral involvement. The mechanism of progression remains incompletely elucidated. Chemokines and their receptors are known to be involved in disease mechanisms, with CXCL12 and CXCR4 playing a critical role in carcinogenesis, invasion and cancer cell migration in various carcinomas. To investigate the expression of CXCL12 and CXCR4 in different cutaneous stages of MF. Formalin-fixed, paraffin-embedded skin samples from 40 patients with MF (21 patch stage, 10 plaque stage, nine tumour stage) and 30 non-neoplastic control skin samples were analysed. CXCL12 and CXCR4 were assessed by quantitative reverse-transcription polymerase chain reaction and immunohistochemical staining. The expression level of mRNA for CXCL12 in plaque-stage MF was significantly higher than in control skin (P = 0.0035), or patch-stage (P = 0.0108) or tumour-stage disease (P = 0.0089). The CXCR4 mRNA expression level in plaque-stage disease was significantly higher than in control skin (P = 0.0090) or patch-stage disease (P = 0.0387). CXCL12- and CXCR4-positive cell rates in patch-stage and plaque-stage MF were significantly higher than those in control skin (P < 0.0001). CXCL12- and CXCR4-positive cell rates in tumour-stage MF were significantly lower than those in patch- and plaque-stage disease (P = 0.0274 and P = 0.0492, respectively). Our data suggest that neoplastic T cells in MF are exposed to the microenvironment, given the abundance of CXCL12 during its progression, and also that neoplastic T cells express CXCR4, especially in the pretumour stage. We reveal that the CXCL12-CXCR4 axis plays a critical role in MF progression.

CXCL12-CXCR4 axis promotes the natural selection of breast cancer cell metastasis.

CXCR4 and its ligand CXCL12 can promote the proliferation, survival, and invasion of cancer cells. They have been shown to play an important role in regulating metastasis of breast cancer to specific organs. High CXCR4 expression was also correlated to poor clinical outcome. Previous study also showed that tumor cells express a high level of CXCR4 and that tumor metastasis target tissues (lung, liver, and bone) express high levels of the ligand CXCL12, allowing tumor cells to directionally migrate to target organs via a CXCL12-CXCR4 chemotactic gradient. However, the exact mechanisms of how CXCR4 and CXCL12 enhance metastasis and/or tumor growth and their full implications on breast cancer progression are unknown. Yet it is likely that chemokine receptor signaling may provide more than just a migrational advantage by also helping the metastasized cells establish and survive in secondary environments. In this study, we investigated CXCR4 and CXCL12 expression in breast cancer and analyzed its association with clinicopathological factors by immunohistochemistry first. Then, we detected the mRNA and protein expression of CXCR4 and CXCL12 in breast cancer cell lines by Western blot and RT-PCR. The MDA-MB-231 has CXCR4 expression and very weak CXCL12 expression. So, we constructed the functional CXCL12 expression in MDA-MB-231 using a gene transfection technique. Further experiments were conducted to evaluate the effect of CXCL12 transfection on the biological behaviors of MDA-MB-231. The cell proliferation of MDA-MB-231–CXCL12 was accessed by MTT assay; the apoptosis was analyzed by an AnnexinV-FITC/propidium iodide double staining of flow cytometry method; and the cell invasive ability was examined by Matrigel invasion assay. Immunohistochemical analysis showed the co-expression of CXCR4 and CXCL12 correlated with lymph node metastasis and TNM stage (p < 0.01). It suggested that the chemokine CXCL12 and its sole ligand CXCR4 play important role in the malignance of breast cancer. To gain a deeper insight into it, we picked CXCR4-expressing cells MDA-MB-231 to be transfected with CXCL12 stably. The decreased cellular proliferation, increased apoptosis, and invasive ability were found in MDA-MB-231 with successful CXCL12 transfection (p < 0.05). Our findings underlined the CXCL12-CXCR4 axis correlated tightly with breast cancer metastasis. CXCL12-CXCR4 axis can increase the invasion and apoptosis of MDA-MB-231 simultaneously. These data strongly support the hypothesis that CXCL12-CXCR4 axis promotes the natural selection of breast cancer cell metastasis. Our findings could have significant implications in terms of breast cancer aggressiveness and the effectiveness of targeting the receptors and downstream signaling pathways for the treatment of breast cancer.

CXCL12–CXCR4 Contributes to the Implication of Bone Marrow in Cancer Metastasis

The CXCL12-CXCR4 axis is postulated to be a key pathway in the interaction between (cancer) stem cells and their surrounding supportive cells in the (cancer) stem cell niche. As the bone marrow constitutes a unique microenvironment for cancer cells, the CXCL12-CXCR4 axis assists the bone marrow in regulating cancer progression. This interaction can be disrupted by CXCR4 antagonists, and this concept is being used clinically to harvest hematopoietic stem/progenitor cells from the bone marrow. The functions of CXCL12-CXCR4 axis in cancer cell-tumor microenvironment interaction and angiogenesis have been recently studied. This review focuses on how CXCL12-CXCR4 helps the bone marrow in creating a tumor mircoenvironment that results in the cancer metastasis. It also discusses ongoing research regarding the clinical feasibility of CXCR4 inhibitors.

Effect of the LPA-mediated CXCL12-CXCR4 axis in the tumor proliferation, migration and invasion of ovarian cancer cell lines.

Ovarian cancer is the most fatal gynecological cancer, with a 5-year survival rate of only 30%. Lysophosphatidic acid (LPA), which possesses growth factor-like functions, is a major regulatory factor in the peritoneal metastasis of ovarian cancer. LPA stimulates the expression of numerous genes that are associated with angiogenesis and metastasis. Ovarian epithelial carcinoma specifically expresses chemotactic factor C-X-C motif chemokine ligand 12 (CXCL12) and its receptor, CXC receptor 4 (CXCR4). The CXCL12-CXCR4 axis directly contributes to ovarian cancer cell proliferation, migration and invasion. The present study investigated the regulation of LPA on the CXCL12-CXCR4 axis and the effect of the LPA-mediated CXCL12-CXCR4 axis on the tumor proliferation, migration and invasion of ovarian cancer cell lines. The CXCR4 proteins expressed in the cell membrane and the cytoplasm of ovarian cancer cells, CAOV3 and SKOV3, were detected by immunocytochemistry. The expression of CXCR4 and CXCL12 was increased in the ovarian cancer cells in a dose- and time-dependent manner when treated with LPA compared with the control groups (P<0.05), as determined by reverse transcription polymerase chain reaction and flow cytometry. LPA (20 μM) and CXCL12 (100 ng/ml) enhanced the proliferation, migration and invasion of the ovarian cancer cells, CAOV3 and SKOV3, as identified by MTT, Transwell and Matrigel assays following co-treatment for 24 h. LPA promoted invasiveness of ovarian cancer by upregulating CXCL12-CXCR4 axis expression.

Inhibiting Metastatic Breast Cancer Cell Migration via the Synergy of Targeted, pH-triggered siRNA Delivery and Chemokine Axis Blockade

Because breast cancer patient survival inversely correlates with metastasis, we engineered vehicles to inhibit both the C-X-C chemokine receptor type 4 (CXCR4) and lipocalin-2 (Lcn2) mediated migratory pathways. pH-responsive liposomes were designed to protect and trigger the release of Lcn2 siRNA. Liposomes were modified with anti-CXCR4 antibodies to target metastatic breast cancer (MBC) cells and block migration along the CXCR4-CXCL12 axis. This synergistic approach—coupling the CXCR4 axis blockade with Lcn2 silencing—significantly reduced migration in triple-negative human breast cancer cells (88% for MDA-MB-436 and 92% for MDA-MB-231). The results suggested that drug delivery vehicles engineered to attack multiple migratory pathways may effectively slow progression of MBC.

Role of the CXCL12–CXCR4 axis in the development of deep rectal endometriosis

Immunological and angiogenetic factors enhance the implantation of endometrial cells in the peritoneal cavity. The aim of this work was to determine the role of the CXCL12–CXCR4 axis in the attraction and the peritoneal implantation of endometriotic stromal cells in deep infiltrating endometriosis (DIE). Biopsies of DIE nodules were obtained from 14 patients undergoing surgical treatment for DIE with low rectal involvement and from 12 patients without macroscopic endometriosis undergoing laparoscopy. CXCR4 expression was evaluated by Western blot analysis and flow cytometry in eutopic endometrial cells and DIE stromal cells in primary cultures derived from the biopsies. CXCL12-induced migration of DIE eutopic endometrial stromal cells was evaluated by transwell migration. CXCL12 was assayed in peritoneal fluids by ELISA. CXCR4 expression was higher in eutopic endometrial stromal cells than in control endometrial cells (p<0.05) and in DIE stromal cells (p<0.05). Eutopic endometrial stromal cells were more attracted by CXCL12 than control cells (p<0.01). CXCL12 was higher in DIE peritoneal fluids than in controls (p<0.05). CXCR4 was down-regulated in deep infiltrating endometriotic stromal cells. The CXCL12–CXCR4 axis plays a role in the attraction of eutopic endometrial cells into the peritoneal cavity, and the down-regulation of CXCR4 in resident endometriotic cells could cause their arrest in situ.

The Effects of Tamoxifen in Combination with Tranilast on CXCL12-CXCR4 Axis and Invasion in Breast Cancer Cell Lines.

It has been reported that CXCL12 binding to CXCR4 induces several intracellular signaling pathways, and enhances survival, proliferation, and migration of malignant cells. In the present study, we examined the effects of anti-estrogen tamoxifen and anti-allergic tranilast drugs as a single or in combination on invasion by two in-vitro invasion assays, wound-healing and matrigel invasion on MCF-7 and MDA-MB-231 human breast cancer cell lines. The mRNA expression levels of CXCR4 and CXCL12 were measured by quantitative real time-RT PCR and CXCL12 protein levels were evaluated by ELISA assay. The data showed that treatment with tamoxifen and tranilast as a single or in combination resulted in decreased CXCR4 and CXCL12 mRNA and CXCL12 protein expression levels. Both in-vitro invasion assays markedly showed synergistic effect of tamoxifen when combined with tranilast drug. Either ER-positive or ER-negative breast cancer cells were sensitive to this combination therapy. In conclusion, Tranilast increases antimetastatic effect of tamoxifen. The synergistic effect of tranilast is not estrogen dependent; however tamoxifen may sensitize the cells for the action of tranilast. The data also support the importance of the CXCR4/CXCL12 interaction in breast cancer metastasis, and further suggest that CXCR4 and CXCL12 are critical targets for tamoxifen and tranilast in combination or alone.

BMS-936564 (Anti-CXCR4 Antibody) Induces Specific Leukemia Cell Mobilization and Objective Clinical Responses In CLL Patients Treated Under a Phase I Clinical Trial

Expression of CXCR4 receptor (CD184) has been reported in different malignancies including Chronic Lymphocytic Leukemia (CLL) and the CXCR4-CXCL12 axis has been described to play a very important role in cancer development. Moreover, this pathway can be inhibited using CXCR4 antagonists.Previously, we have shown that BMS-936564, an anti-CXCR4 IgG4 antibody, can block in vitro the CXCR4/CXCL12 pathway in CLL overcoming stromal cell protection by inducing apoptosis, inhibiting F-actin polymerization, and migration of cells. These findings provide the rationale for an ongoing phase I clinical study in which CLL subjects received four weekly infusions of BMS-936564 antibody followed by 5 monthly cycles of bendamustine, rituximab and BMS-936564 antibody (BRB chemoimmunotherapy). Eleven (n=11) subjects with relapse / refractory disease have been enrolled and treated in this study. Here, we present correlative studies performed in three of those CLL subjects with samples collected at different time points (day 1, 2, 8, 15 and 22) during the monotherapy cycle of BMS-936564.All three subjects showed leukocytosis that was detected as early as in 4 hours following the initial BMS-936564 infusion (median increase of 64.6% above base line; range: 59.7% - 112.7%). Leukocytosis was present during the entire four weeks of monotherapy with BMS-936564. Leukocytosis in these three patients was due primarily to increase in absolute counts of CLL cells (Median increase of 129.6%; range: 95.3% - 324.8%). Interestingly, there was no evidence of increase in the absolute number of normal lymphocytes. Only one of the three patients showed an increase in neutrophil counts after infusion of BMS-936564.We observed changes in the level of CXCR4 expression after infusion with BMS-936564. All subjects showed CXCR4 down-regulation in peripheral CLL cells with a median percentage decrease in the level of expression of 106.7% (Range: 25.1% - 350.7%). We did not observe changes in CXCR4 expression in normal B cells.In contrast to our in vitro studies where we observed that all tested CLL samples underwent apoptosis after treatment with BMS-936564, only one subject showed significant increase in the percentage of apoptosis after infusion of BMS-936564 in vivo. The levels of apoptosis detected were relatively low (<18%) and this suggests a potential mechanism of rapid clearance of apoptotic cells.Importantly, associated with the mobilization of CLL cells, all three patients showed significant decrease in the lymphadenopathy size with a median decrease of 59.83% (Range: 55.6-72%). Lymphadenopathy response was assessed using stringent IWCLL response assessment criteria by an independent radiologist using CT scan measurements.All three subjects tolerated the infusions well with no evidence of side effects or toxicities. After the initial monotherapy treatment phase with BMS-936564 antibody, all three subjects received BRB chemoimmunotherapy combination and had a dramatic decrease in lymphocytosis during the first week after initiation of combination therapy. These subjects are currently finishing treatment under protocol and response assessments will be presented during the meeting.In conclusion, we found that BMS-936564 infusion in subjects with CLL induces specific leukemia cell mobilization associated with an objective decrease in the lymphadenopathy bulk and phenotypic changes associated with CXCR4 down-regulation in peripheral leukemia cells. These findings strongly suggest a CXCR4-CXCL12 dependent mobilization process of CLL cells from lymphatic tissues. Together, our study provides for the first time evidence at in vivo level, which supports inhibition of the CXCR4-CXCL12 pathway mediated by BMS-936564 in CLL patients. These findings suggest a compartmental biological distribution and recirculation of CLL cells opening the possibility to explore targeted therapies against minimal residual disease that traditionally has been refractory to conventional chemoimmunotherapy treatment and contributes to the incurable characteristics of this disease. Disclosures:Kuhne:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Sabbatini:Bristol-Myers Squibb: Employment. Kipps:Bristol-Myers Squibb: Research Funding. Cardarelli:Bristol-Myers Squibb: Employment.

Neutrophil mobilization via plerixafor-mediated CXCR4 inhibition arises from lung demargination and blockade of neutrophil homing to the bone marrow

Blood neutrophil homeostasis is essential for successful host defense against invading pathogens. Circulating neutrophil counts are positively regulated by CXCR2 signaling and negatively regulated by the CXCR4-CXCL12 axis. In particular, G-CSF, a known CXCR2 signaler, and plerixafor, a CXCR4 antagonist, have both been shown to correct neutropenia in human patients. G-CSF directly induces neutrophil mobilization from the bone marrow (BM) into the blood, but the mechanisms underlying plerixafor-induced neutrophilia remain poorly defined. Using a combination of intravital multiphoton microscopy, genetically modified mice and novel in vivo homing assays, we demonstrate that G-CSF and plerixafor work through distinct mechanisms. In contrast to G-CSF, CXCR4 inhibition via plerixafor does not result in neutrophil mobilization from the BM. Instead, plerixafor augments the frequency of circulating neutrophils through their release from the marginated pool present in the lung, while simultaneously preventing neutrophil return to the BM. Our study demonstrates for the first time that drastic changes in blood neutrophils can originate from alternative reservoirs other than the BM, while implicating a role for CXCR4-CXCL12 interactions in regulating lung neutrophil margination. Collectively, our data provides valuable insights into the fundamental regulation of neutrophil homeostasis, which may lead to the development of improved treatment regimens for neutropenic patients.

CXCL12-CXCR4 Promotes Proliferation and Invasion of Pancreatic Cancer Cells

CXCL12 exerts a wide variety of chemotactic effects on cells. Evidence indicates that CXCL12, in conjunction with its receptor, CXCR4, promotes invasion and metastasis of tumor cells. Our objective was to explore whether the CXCL12-CXCR4 biological axis might influence biological behavior of pancreatic cancer cells. Miapaca-2 human pancreatic cancer cells were cultured under three different conditions: normal medium (control), medium + recombinant CXCL12 (CXCL12 group), or medium + CXCR4-inhibitor AMD3100 (AMD3100 group). RT-PCR was applied to detect mRNA expression levels of CXCL12, CXCR4, matrix metalloproteinase 2 (MMP-2), MMP-9, and human urokinase plasminogen activator (uPA). Additionally, cell proliferation and invasion were performed using CCK-8 colorimetry and transwell invasion assays, respectively. CXCL12 was not expressed in Miapaca-2 cells, but CXCR4 was detected, indicating that these cells are capable of receiving signals from CXCL12. Expression of extracellular matrix-degrading enzymes MMP-2, MMP- 9, and uPA was upregulated in cells exposed to exogenous CXCL12 (P<0.05). Additionally, both proliferation and invasion of pancreatic cancer cells were enhanced in the presence of exogenous CXCL12, but AMD3100 intervention effectively inhibited these processes (P<0.05). The CXCL12-CXCR4 biological axis plays an important role in promoting proliferation and invasion of pancreatic cancer cells.

CXCL14 is a natural inhibitor of the CXCL12–CXCR4 signaling axis

Activation of the CXCL12–CXCR4 pathway is crucial for the migration of hematopoietic stem cells, various immune cells, and malignant tumor cells. Here, we show that another CXC chemokine, CXCL14, specifically binds to CXCR4 with high affinity and inhibits the CXCL12-mediated chemotaxis of human leukemia-derived cell lines and CD34+ hematopoietic progenitor cells. Thus, CXCL14 functions as a natural inhibitor of CXCL12. Our observations suggest that CXCL14 represents, along with CXCR7, molecules that co-evolved with the CXCL12–CXCR4 axis to modulate important physiological processes in development, stem cell maintenance, and immune responses. Structured summary of protein interactionsCXCR4physically interacts with CXCL14anti bait coimmunoprecipitation by (View interaction).

Abstract 5032: Elucidating the role of CXCR4 in Ewing sarcoma.

Abstract Background: Ewing sarcoma (ES) is the second most common malignant bone tumor in pediatric patients. While intensification of chemotherapy has improved the outlook for patients with localized disease, little progress has been made for those with metastatic or relapsed disease. Prognosis for these patients remains dismal with fewer than 20% surviving. Moving forward, we need to develop molecularly targeted therapy for patients with high-risk disease. The mechanisms underlying metastatic relapse in ES remain unknown but gene encoding the chemokine receptor CXCR4 has been shown to be upregulated in metastatic ES. The CXCL12-CXCR4 axis contributes to tumor metastasis in other sarcomas but its function in ES is not yet known. Objective: In the current study we are investigating whether CXCR4 is a mediator of ES cell metastasis and may be useful as a biomarker of high-risk disease. Results: Using qRT-PCR and flow cytometry we have shown that expression of CXCR4 transcript and protein varies significantly across ES cell lines. Importantly, the frequency of CXCR4+ cells and CXCR4 expression levels were found to be highly dynamic and were altered under different experimental conditions. Specifically, both serum deprivation and hypoxia induced upregulation of CXCR4 in TC32, TC71, and A673 ES cells. CXCR4+ cell frequency was found to be consistently highest in TC32 where the frequency ranged from 35% in ambient conditions to 62% in serum deprivation. When sorted into CXCR4+ and CXCR4- populations TC32 populations returned to their baseline state of 40% CXCR4+ cells within 3 weeks indicating that CXCR4+ cells can change into CXCR4- cells and vice versa. Interestingly, CXCR4 transcript was identified to be increased in slowly proliferating (PKH-dye-retaining) TC71 and A673 cells compared to more rapidly dividing counterparts. These data implicate CXCR4 as a potential marker of putative cancer stem cells. Finally, initial immunohistochemical studies have demonstrated highly variable patterns of CXCR4 expression in primary ES tumor samples. Summary: ES cells express CXCR4 both in primary tumors and in cultured cell lines. However, the expression is both variable and highly dynamic. Specifically, we have found that ES cells can switch back and forth between CXCR4- and CXCR4+ states in response to environmental cues including serum deprivation and hypoxia. Ongoing work is now testing the hypothesis that CXCR4+ ES cells are more migratory than CXCR4- cells and may thus be a source of metastasis. To further test this hypothesis we are using correlative studies of clinically annotated tumor microarrays to determine if a high frequency of CXCR4+ cells at diagnosis is associated with an increased risk of metastatic relapse. Citation Format: Melanie A. Krook, Lauren A. Nicholls, Natashay J. Bailey, Amy Han, Chris Scannell, Dafydd Thomas, Elizabeth R. Lawlor. Elucidating the role of CXCR4 in Ewing sarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5032. doi:10.1158/1538-7445.AM2013-5032