Presence Of CXCL12 Research Articles

P1399: EXTRAMEDULLARY HEMATOPOIESIS IN THE ADULT ADRENAL GLAND AS A MODEL OF THE MINIMAL HEMATOPOIETIC NICHE

Background: Hematopoietic Stem and Progenitor Cells (HSPCs) reside in the hematopoietic niche, a structure that regulates the balance of cellular quiescence, self-renewal and commitment towards progenitor cells in a demand-adapted manner. The bone marrow (BM) hematopoietic niche is formed by several cellular players, mainly endothelial cells, osteoblasts, adipocytes, and a variety of stromal cells. While the BM niche contains a complex stromal population, evidence exists for simpler, albeit functional, extramedullary hematopoietic (EMH) niches. EMH can be found associated to adipose tissue. An example of adipose-associated EMH is myelolipoma, an adrenal tumor composed of myeloid and adipose tissue. Myelolipoma is frequent in patients with congenital adrenal hyperplasia, an endocrine disorder that associates an increase in ACTH. Aims: We aimed to produce an EMH animal model that would recapitulate myelolipoma to study a minimalistic, boneless hematopoietic niche. Methods: We designed a strategy to induce EMH in the adrenal gland. For this, we injected hematopoietic cytokines (Granulocyte colony-stimulating factor (G-CSF)) and pituitary axis hormones (ACTH) as well as androgens (testosterone) in splenectomized C57BL/6J mice and studied the hematopoietic-supportive capacity of the induced adrenal glands. Results: Here, we show that the adult adrenal gland can be transformed into a hematopoietic supportive environment. Upon hormonal stimulation, the adult adrenal gland can be induced to recruit and host hematopoietic cells, including serially transplantable HSCPs. The adrenal stroma supports HSPC proliferation and function; furthermore, the adrenal stroma contains a CXCL12+ population, reminiscent of BM CXCL12-Abundant Reticular (CAR). These findings are mirrored by the presence of CXCL12+ cells in human myelolipoma samples, a benign adrenal tumor composed of adipose and hematopoietic tissue. Summary/Conclusion: We present our model as a novel tool to increase our understanding of the physiology of hematopoietic support and to facilitate the development of a boneless niche model.

Rejuvenating Effector/Exhausted CAR T Cells to Stem Cell Memory-Like CAR T Cells By Resting Them in the Presence of CXCL12 and the NOTCH Ligand.

Resting CAR T cells with our defined factors reprograms exhausted state to SCM-like state and enables development of improved CAR T-cell therapy.

MAPK-Erk1/2 Pathway Activation and Survival Of Chronic Lymphocytic Leukemia (CLL) Cells Is Induced By In Vitro Modeling Of The Tumor Microenvironment and Can Be Effectively Inhibited Using The MEK1 Inhibitor MEK162

Infiltration of chronic lymphocytic leukemia (CLL) cells into the lymph node and bone marrow microenvironments promotes leukemic cell survival, proliferation and ultimately drives disease progression. Successful treatment of CLL relies on targeting the cells within these niches.Activity of the MAPK-Erk1/2 pathway is considered to be crucial for the survival of a diverse range of cancer cell types, including CLL cells. However, to date pre-clinical studies of inhibitors of MEK1/2, a key component in this pathway, have demonstrated little efficacy against CLL cells.We have recently shown that MAPK-Erk1/2 pathway activity is attenuated during ex vivo cell processing and subsequent culture of primary CLL cells; an observation which suggests that factors exist in vivo that maintain Erk1/2 activity and promote cell survival.Here we present data that show that in vitro modeling of the effects of the microenvironment on primary CLL cells, using recombinant stroma-derived factor CXCL12 or media conditioned by the HS5 stromal cell line (HS5-CM), results in a significant increase in MAPK-Erk1/2 pathway activity. Treatment with the MEK1/2 inhibitor MEK162 (Novartis) blocked the effects of CXCL12 and HS5-CM on Erk1/2 activation in CLL cells. In the presence of CXCL12 or HS5-CM, MEK162 induced apoptosis in a significantly (P< 0.05) greater proportion of cells compared to treatment of cells cultured in media alone (media: 30.14 ± 5.74%, CXCL12: 78.34 ± 9.06%, HS5 CM: 65.79 ± 12.88%). Furthermore, MEK162 completely abrogated the survival advantage conferred by the presence of HS5-CM and reduced cell migration through a permeable support towards HS5-CM by an average of 35.64 +/- 8.48%.In conclusion, our work suggests that MAPK-Erk1/2 signaling is abrogated in CLL cells during ex vivo processing and subsequent culture. This observation provides a rational explanation for the current lack of in vitro data concerning any efficacy of MEK inhibitors against CLL cells. Our data show that by introducing purified stroma-derived factor (SDF-1a/CXCL12) or media conditioned by the HS5 stromal cell line it is possible to recapitulate the activity of Erk1/2 and increase CLL-cell sensitivity to the MEK1/2 inhibitor MEK162. These observations highlight the importance of modeling the influence of the microenvironment in all pre-clinical drug-efficacy studies in CLL and suggest that inhibition of MEK1/2 may represent a novel treatment strategy for CLL. Disclosures:No relevant conflicts of interest to declare.

High-Throughput Screening of a Functional Human CXCL12-CXCR4 Signaling Axis in a Genetically Modified S. cerevisiae: Discovery of a Novel Up-Regulator of CXCR4 Activity.

CXCL12 activates CXCR4 and is involved in embryogenesis, hematopoiesis, and angiogenesis. It has pathological roles in HIV-1, WHIM disease, cancer, and autoimmune diseases. An antagonist, AMD3100, is used for the release of CD34+ hematopoietic stem cells from the bone marrow for autologous transplantation for lymphoma or multiple myeloma patients. Adverse effects are tolerated due to its short-term treatment, but AMD3100 is cardiotoxic in clinical studies for HIV-1. In an effort to determine whether Saccharomyces cerevisiae expressing a functional human CXCR4 could be used as a platform for identifying a ligand from a library of less ∼1,000 compounds, a high-throughput screening was developed. We report that 2-carboxyphenyl phosphate (fosfosal) up-regulates CXCR4 activation only in the presence of CXCL12. This is the first identification of a compound that increases CXCR4 activity by any mechanism. We mapped the fosfosal binding site on CXCL12, described its mechanism of action, and studied its chemical components, salicylate and phosphate, to conclude that they synergize to achieve the functional effect.

CORRIGENDUM.

CorrigendumCORRIGENDUMPublished Online:05 Apr 2019https://doi.org/10.1152/ajpcell.zh0-8419-corr.2019Original articleMoreSectionsPDF (593 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Lear T, Dunn SR, McKelvey AC, Mir A, Evankovich J, Chen BB, Liu Y. RING finger protein 113A regulates C-X-C chemokine receptor type 4 stability and signaling. Am J Physiol Cell Physiol 313: C584–C592, 2017. First published October 4, 2017; doi:10.1152/ajpcell.00193.2017.— In Fig. 4, the β-actin blots presented in A and B were duplicated in error. Figure 4A has been corrected and the corrected figure is shown below. The changes made do not affect the conclusions presented in this study.Fig. 4.RNF113A regulates CXCR4 signaling and cellular motility. A: HeLa cells were transfected with RNF113A-V5 before CXCL12 treatment and immunoblotting for phosphorylated kinases. Densitometry relative to Empty at time 0 is shown beneath immunoblots. B: HeLa cells were transfected with plasmid encoding shRNA targeting RNF113A before treatment with CXCL12 (6 nM) for the indicated times. Cells were collected and immunoblotted for activation of downstream CXCR4 signalers. Densitometry relative to Ctrl shRNA at time 0 is shown beneath immunoblots. C and D: wound healing assay. HeLa cells were transfected with empty or RNF113A-encoded plasmids, or with plasmids encoding shRNA against scramble or RNF113A and allowed to grow to 80–90% confluency before starvation and wounding with pipette tip. Extent of gap closure was measured at 48 or 72 h and in the absence or presence of CXCL12. Gap closure is quantified below. Data represent mean values ± SE (n = 3–4 per group; *P < 0.05, significant between indicated groups, NS, P > 0.05, not significant between indicated groups, two-sided t-test). “KD” refers to shRNA.Download figureDownload PowerPointThis article has no references to display. Download PDF Previous Back to Top FiguresReferencesRelatedInformation Related ArticlesRING finger protein 113A regulates C-X-C chemokine receptor type 4 stability and signaling 07 Nov 2017American Journal of Physiology-Cell Physiology More from this issue > Volume 316Issue 4April 2019Pages C582-C582 Copyright & PermissionsCopyright © 2019 the American Physiological Societyhttps://doi.org/10.1152/ajpcell.zh0-8419-corr.2019PubMed30951388History Published online 5 April 2019 Published in print 1 April 2019 Metrics

CXCL12 modulates the radiosensitivity of cervical cancer by regulating CD44.

The aim of the present study was to investigate the regulation of stromal cell‑derived factor 1 (CXCL12) in the radioresistance of cervical cancer, which was upregulated in tumors in our previous study. A CCK‑8 assay was used to detect cell viability. Flow cytometry was used to measure cell apoptosis and the expression levels of CD44 and CXCR4. ELISA was performed to measure the expression level of CXCL12 protein and CXCL12 mRNA was detected by reverse transcription‑quantitative polymerase chain reaction assays. Cell viability and apoptosis were determined with or without treatment with CXCL12 small interfering (si)RNA to examine the function of CXCL12 in Hela cells. The expression level of CD44 antigen (CD44) and C‑X‑C chemokine receptor type 4 (CXCR4) were measured using flow cytometry in the presence of CXCL12 and irradiation. In the present study, it was demonstrated that inhibition of CXCL12 reduced cell viability and increased cellular apoptosis in Hela cells treated with irradiation. Following treatment with CXCL12 siRNA, the expression level of CD44 was downregulated and the expression level of CXCR4 was upregulated. This effect of regulation additionally occurred in the presence of irradiation. In conclusion, the present data demonstrated that CXCL12 served an important role in the radioresistance of cervical cancer, suggestinh a novel therapeutic target.

The Bruton's Tyrosine Kinase (BTK) Inhibitor ARQ 531 Effectively Inhibits Wild Type and C481S Mutant BTK and Is Superior to Ibrutinib in a Mouse Model of Chronic Lymphocytic Leukemia

Introduction: The Bruton's tyrosine kinase inhibitor ibrutinib improves survival in chronic lymphocytic leukemia (CLL) compared to standard chemotherapy or immune therapy. In a subset of patients, somatic mutation (C481S) of its binding site results in acquired resistance to ibrutinib therapy with poor clinical outcome. ARQ 531 is an ATP competitive, orally bioavailable, potent inhibitor of BTK and other relevant kinases including the majority of Src and Tec family kinases. Herein we present preclinical data with ARQ 531 in CLL including C481S mutated BTK and its efficacy versus ibrutinib in the TCL1 mouse model of CLL.Methods: Potency of ARQ 531 and its binding kinetics were measured in enzymatic and Surface Plasmon Resonance (SPR) binding assays. Primary CLL B cells were negatively selected and treated with 1μM ARQ 531. BCR signaling was investigated by immunoblot following a 1 hour drug incubation. CLL cells migrating towards CXCL12 and CXCL13 after 4 hours across a 5.0 micron transwell insert were counted by flow cytometry. Annexin V and propidium iodide flow cytometry was used to measure CLL viability over a range of drug concentrations and time. CpG mediated CLL cell activation was measured by CD40 and CD86 expression by flow cytometry. In vivo investigation utilized B6 mice engrafted with 1E7 CD5+/CD19+ TCL1 lymphocytes via tail vein injection. Mice were randomized to treatment with vehicle, ARQ 531, or ibrutinib following the establishment of a CD5+/CD19+ population >10% in peripheral blood.Results: At 72 hours the viability of CLL cells treated with 0.1µM, 1.0µM, or 10.0µM ARQ 531 was found to be 94%, 67%, and 50% that of untreated samples, respectively (p=0.76, p<0.001, p=0.016) in comparison to the 71% relative viability seen with 1µM ibrutinib (p<0.001). ARQ 531 demonstrates dose dependent inhibition of BTK, AKT, and ERK phosphorylation. Stimulation through TLR9 by CpG ligand increases expression of the activation markers CD40 and CD86; ARQ 531 decreased CpG induced expression of CD40 by 25% and CD86 by 40% (p=0.022, p=0.041). ARQ 531 in the presence of CXCL12 or CXCL13 decreased migration by 51% and 66%, respectively (p=0.015, p=0.025), below baseline migration of untreated CLL.Unlike ibrutinib, ARQ 531 inhibits both wild type and C481S BTK with an IC50 less than 1nM in a biochemical assay, and inhibits phosphorylation of C481S mutated BTK in LV125 transfected HEK293T cells. Cytotoxicity was also observed in CLL cells isolated from patients with C481S BTK mutations, where the viability of cells treated with 1µM ARQ 531 was 72% that of untreated cells at 72 hours (p=0.038). SPR binding assay showed a residence time of 51 minutes in wild type BTK and 56 minutes in C481S mutated BTK.The TCL1 mouse model displays active BCR signaling and responds to treatment with the BTK inhibitors ibrutinib and acalabrutinib (Ponader S, 2012; Herman SEM, 2015). Following establishment of leukemia in the TCL1 transfer model, we randomized mice to treatment with vehicle (n=14), 25 mg/kg ibrutinib (n=6), 50 mg/kg ARQ 531 (n=14) or 75 mg/kg ARQ 531 (n=14) given by daily gavage. ARQ 531 significantly improved survival over both ibrutinib and vehicle (median survival: 36 days vehicle, 53 days ibrutinib, not reached by 74 days 50mg/kg and 75 mg/kg ARQ 531, p<0.0001). Mice receiving ARQ 531 have consistently lower lymphocyte counts on microscopic examination and reduced CD5+/CD19+ populations via flow cytometry compared to vehicle or ibrutinib. Additionally, we have observed increases in absolute neutrophil count over time in mice receiving ARQ 531. Pharmacokinetic studies are pending; however, in a single oral dose study of 10mg/kg in monkeys, the bioavailability was 72.4% with a CMax of 9µM and a half-life greater than 24 hours.Conclusion: The BTK inhibitor ARQ 531 is a potent inhibitor of BTK with promising activity both in vitro and in vivo. Multi-targeted inhibition of cytokine, chemokine, and BCR pathways by ARQ 531 decreases activation, migration, and viability of CLL cells. Unlike ibrutinib, ARQ 531 inhibits activation of C481S mutated BTK variants and maintains cytotoxicity in ibrutinib resistant clones. Additionally, ARQ 531 has demonstrated remarkable efficacy in an in vivo TCL1 adoptive transfer model, improving survival to a greater extent than ibrutinib and potentially restoring granulocyte production. These data justify continued preclinical work and development to facilitate transition to clinical trials. DisclosuresEathiraj:ArQule, Inc: Employment. Abbadessa:ArQule, Inc: Employment. Schwartz:ArQule, Inc: Employment. Woyach:Acerta: Research Funding; Morphosys: Research Funding; Karyopharm: Research Funding.

Downregulation of CXCL12 in mesenchymal stromal cells by TGF\u03b2 promotes breast cancer metastasis

Mesenchymal stromal cells (MSCs) are one of major components of the tumour microenvironment. Recent studies have shown that MSC tumour residence and their close interactions with inflammatory factors are important factors that affect tumour progression. Among tumour-associated inflammatory factors, transforming growth factor β (TGFβ) is regarded as a key determinant of malignancy. By employing a lung metastasis model of a murine breast cancer, we show here that the prometastatic effect of MSCs was dependent on their response to TGFβ. Interestingly, we found that MSC-produced CXCL12, an important chemokine in tumour metastasis, was markedly inhibited by TGFβ. Furthermore, silencing of CXCL12 in TGFβ-unresponsive MSCs restored their ability to promote tumour metastasis. We found that 4T1 breast cancer cells expressed high levels of CXCR7, but not of CXCR4, both of which are CXCL12 receptors. In presence of CXCL12, CXCR7 expression on tumour cells was decreased. Indeed, when CXCR7 was silenced in breast cancer cells, their metastatic ability was inhibited. Therefore, our data demonstrated that sustained expression of CXCL12 by MSCs in the primary tumour site inhibits metastasis through reduction of CXCR7, while, in the presence of TGFβ, this CXCL12 effect of MSCs on tumour cells is relieved. Importantly, elevated CXCR7 and depressed CXCL12 expression levels were prominent features of clinical breast cancer lesions and were related significantly with poor survival. Our findings reveal a novel mechanism of MSC effects on malignant cells through which crosstalk between MSCs and TGFβ regulates tumour metastasis.

Abstract 1453: CXCR4 and CXCR7 homodimers and heterodimers play differential roles in breast cancer

Abstract The majority of breast cancer deaths are due to the metastasis of tumor cells to secondary organs. One of the most studied metastatic processes is the migration of chemokine receptor-expressing cancer cells towards their ligands in other organs, a mechanism where the receptor CXCR4 and the chemokine CXCL12 play key roles. This process, however, may be affected by the expression of the atypical receptor CXCR7, which has recently been linked to breast cancer progression. Indeed, staining of CXCR7 in patient samples is strongly positive in both lobular and ductal breast cancers. Given CXCR7's ability to form heterodimers with CXCR4, we investigated how dual expression of both receptors differs from their lone expression in terms of their signaling and internalization pathways. In this study we created stable single CXCR4 or CXCR7 CHO transfectants or double CXCR4-CXCR7 transient transfectants to examine ligand-dependent receptor internalization. The presence of heterodimers was evaluated using fluorescence resonance energy transfer (FRET). We found that in CHO-CXCR4 the receptor was degraded after a 30 minute stimulation with 10 nM CXCL12, whilst in CHO-CXCR7 the receptor returned to the surface in under 2 hours, reaching higher CXCR7 levels than before treatment. RT-PCR data suggested that this increase was partially due to induction of further CXCR7 expression. When the receptors were coexpressed, a similar pattern could be seen, although the CXCR7 recycling levels were not as high. CXCR4 degradation could be partially inhibited by pretreating cells with 1 μM lactacystin, a proteasome inhibitor. When cells were stimulated for 1 hour with 1 nM VUF11207, a CXCR7 agonist, no significant effect was seen on CHO-CXCR4 whilst CHO-CXCR7 cells showed internalization and recycling. Supporting RT-PCR data showed that the agonist did not trigger CXCR7 expression like seen with CXCL12. Interestingly, when coexpressed, both CXCR4 and CXCR7 showed degradation after internalization. Similar results were observed with MDA-MB-231 cells transfected with CXCR4 or CXCR7. The effects of the binding were also assessed by measuring ERK phosphorylation by Western blot and cell-based ELISA. An early but transient (5-15 minutes) ERK activation was seen in CHO-CXCR4 cells, whilst CHO-CXCR7 cells showed a sustained activation of up to 2 hours. On the other hand, CHO-CXCR4-CXCR7 cells showed a very strong phosphorylation at 5 minutes that quickly diminished. The role of the receptors in invasion and migration in the presence of CXCL12 was also assessed. In summary, this study highlights how CXCR4/CXCR7 heterodimerization can create a distinct signaling entity with unique properties and alter receptor pharmacology. This could be used to create therapeutic compounds to reduce breast cancer cell migration. Citation Format: Irene del Molino del Barrio, Simi Ali, John Kirby, Annette Meeson. CXCR4 and CXCR7 homodimers and heterodimers play differential roles in breast cancer. [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 1453.

Abstract 4077: Crucial factors of the inflammatory microenvironment (IL-1β/TNF-α/TIMP-1) promote the selection of highly malignant hemopoietic clone of myelofibrosis

Abstract Myelofibrosis (MF) is a clonal disorder of the hemopoietic stem/progenitor cells (HSPCs). Together with molecular abnormalities (mutations in JAK2, Calreticulin (CALR) and MPL genes), chronic inflammation is the major hallmark of MF pathogenesis. It is argued that the up-regulated production of pro-inflammatory cytokines selects for the malignant clone. However, the key players linking inflammation and cancer in MF are still poorly known. Here, we investigated the in vitro effects of crucial factors of the inflammatory microenvironment (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, Tissue Inhibitor of Metalloproteinases (TIMP)-1 and ATP) on the functional behaviour of MF-derived HSPCs. Plasma TIMP-1, TNF-α and IL-1β were measured by ELISA assay. Circulating CD34+ cells from MF patients (JAK2V617F (10 cases) and CALR (10 cases) mutations) or cord blood (CB; 8 samples) were functionally characterized after incubation with or without ATP (1000 μM), IL-1β (10 ng/mL), TNF-α (10 ng/mL) or TIMP-1 (100 ng/mL) (alone or in combination). Cells were then analyzed for survival/apoptosis (Annexin-V/Propidium Iodide staining), cell cycle and clonogenic capacity. Migration was assessed towards a C-X-C motif chemokine 12 (CXCL12) gradient in the presence or absence of the pro-inflammatory factors. MF-derived CD34+ cells were co-cultured with normal mesenchymal stromal cells (MSCs) in the presence of the pro-inflammatory factors and then evaluated for survival. Regardless mutation status, the plasma levels of IL-1β, TNF-α and TIMP-1 are increased in MF patients and the presence of pro-inflammatory cytokines confers a survival advantage of MF-derived CD34+/CD34+CD38- cells. In addition, MF-derived CD34+ cells promoted cell-cycle progression to the S-phase. Whereas in the JAK2V617F mutated group, the addition of IL-1β or TNF-α ± TIMP-1 impaired the erythroid clonogenic output of the CALR mutated patients. Of note, IL-1β + TNF-α ± TIMP-1 highly promoted the in vitro migration of MF-derived CD34+ cells in the presence of CXCL12. Intriguingly, MF-derived CD34+ cells revealed increased clonogenic ability after migration toward CXCL12 and IL-1β + TNF-α ± TIMP-1, suggesting a pivotal role of inflammation in the selection of the malignant clone. Finally, co-cultures of normal MSCs with MF-derived CD34+ cells sustained the survival effects of IL-1β+TNF-α±TIMP-1. Overall, our data indicate that the behavior of the MF-derived HSPCs can be influenced by regulatory signals provided by the microenvironment through the cooperation between various pro-inflammatory factors. The interplay of these pro-inflammatory factors promotes and selects the circulating MF-derived HSPCs with higher proliferative activity, clonogenic potential and migration ability. Targeting these micro-environmental interactions may be a clinically relevant approach. Citation Format: Daria Sollazzo, Dorian Forte, Nicola Polverelli, Marco Romano, Margherita Perricone, Sofia Fatica, Francesco Lapi, Emanuela Ottaviani, Martinelli Giovanni, Nicola Vianelli, Michele Cavo, Maria Pantaleo, Francesca Palandri, Lucia Catani. Crucial factors of the inflammatory microenvironment (IL-1β/TNF-α/TIMP-1) promote the selection of highly malignant hemopoietic clone of myelofibrosis. [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 4077.

Arhgap21+/- Mice Show Impaired Adhesion, Migration, Homing and Short Term Reconstitution of Hematopoietic Progenitor Cells

The main signaling pathway involved in migration, adhesion and homing of hematopoietic progenitor cells (HPC) is the CXCL-12, chemokine produced by bone marrow (BM) stromal cells that bind to its main receptor CXCR-4, expressed by HPC. ARHGAP21 is a RhoGAP member, negative regulators of RhoGTPAses. It was described that ARHGAP21 is involved in cellular migration and adhesion but its role in hematopoiesis is unknown.Researching Arhgap21 role in hematopoiesis, Arhgap21+/- mice were generated using Embryonic Stem cell obtained from GeneTrap consortium. Hematologic parameters were investigated by blood cell count, colony formation assay in methylcellulose medium and immunophenotipic characterization of hematopoietic populations. Transwell migration and adhesion of Lin- cells assay in the presence of CXCL12 were also performed.In vivo analysis of Arhgap21+/- HPC was achieved with homing and CFU-S assays in sublethally irradiated mice and hematopoiesis stress was generated by 5-fluoracil treatment.Arhgap21+/- mice presented more than 50% reduction in Arhgap21 expression (0.47 ± 0.13) in BM when compared to WT (1 ± 0.03) and live in normal conditions. On the other hand, Arhgap21 mice showed a reduction in BFU-E colonies and erythroid (Terr119+) committed cells in BM together with decreased peripheral blood (PB) Terr119+ cells and red blood cell number and increased medium corpuscular volume.In myeloid compartment, Arhgap21+/- mice presented less GM colonies and myeloid (Gr1/Mac1+) cells in BM followed by an increase of these cells in PB suggesting myeloid mobilization. Corroborating to this, whenArhgap21+/- BM was challenged for hematopoiesis stress with 5FU, the animals showed increased neutrophil number in peripheral blood 14 days (WT: 1887/mm3 ±721.9; Arhgap21+/-:3325/mm3 ±1640, p=0.02) and 21days (WT: 1264/mm3 ±635; Arhgap21+/-: 2182/mm3 ±854, p=0.01) after treatment, together with increased number of LSK cells in the BM (WT: 1.3% ±0.4; Arhgap21+/-: 1.9% ±0.3, p=0.006) 28 days after 5FU infusion.Erythroid and myeloid compartments reductions were observed together with an increase of Arhgap21+/- BM short term LSK( Arhgap21+/- : 0.34% ±0.13; WT: 0.18% ±0.07, p=0.03) and long term LSK (Arhgap21+/-: 0.003% ±0.001; WT: 0.002 ±0.0007, p=0.02) suggesting an attempt to restore normal hematopoietic levels.Arhgap21+/- HPC showed reduced CXCL-12-induced migration compared to WT (WT: 100%, Arhgap21+/-: 54.73% ± 13.57, p=0.01) in addition to decreased adhesion to fibronectin (Arhgap21+/-: 15% ± 3.8; WT: 27% ± 3.5, p=0.003) and a4b1 integrin expression (WT: 83.76 ± 4.35; Arhgap21+/-: 71.06 ± 7.00, p=0.008).In homing assay, the percentage of donor Lin- HPC from Arhgap21+/- mice that homed to BM (6.32 ± 2.41) or spleen (3.48 ± 1.57), were lower than those from WT mice (BM=10.09 ± 1.81;p=0.004; spleen=6.9 ± 1.48; p=0.0007) together with higher frequency of these cells in peripheral blood (WT: 6.39 ± 3.38; Arhgap21+/-: 13.95 ± 5.33, p=0.003), suggesting a retention of Arhgap21+/- HPC in the bloodstream, which inefficiently home to BM and spleen. Arhgap21+/- CFU-S capacity decline was also observed (Arhgap21+/-: 20.86 ± 2; WT: 29.29 ± 5.4 p=0.002).This is the first study showing that ARHGAP21 is involved in hematopoiesis and also shows that ARHGAP21 is an important protein for chemotaxis, adhesion, homing and short term reconstitution of HPC.TableHematological ParametersWTArhgap21+/-Hemoglobin (g/dL) *16.19 ± 0.3515.62±0.32RBC (10^6/uL) *10.91 ± 0.310.46±0.25Hematocrit (%)54.78 ± 1.653.75± 1.3MCV (fL) *50 ±0.450.08±0.74WBC (10^3/Ul) *10.08 ±212.9±1.8Platelets(10^3/uL)1793 ±114.31770±96.5Myeloid, Gr1+Mac1+ in BM (%) *47.27 ±4.740.88 ±6.23Erythroid, Terr119+ in BM (%) *9.98 ±4.16.4 ±1.5Short Term HSC, LSK in BM (%) *0.18 ±0.070.34 ±0.13Long Term HSC, LSK CD150+CD48- in BM (%) *0.002 ±0.00070.003 ±0.001Myeloid, Gr1+Mac1+ in PB (%) *7.7 ±1.513.1 ±2.6Erythroid, Terr119+ in PB (%) *38.7 ±4.428.4 ±8.7Myeloid Colonies, CFU-GM (number)66.7 ±14.748.07 ±12.5Erythroid Colonies, BFU-E (number)5.5 ±2.23.2 ±1.5Blood samples were collected from 9 WT and 8 Arhgap21+/- mice. RBC: red blood cell count; MCV: mean cell volume; WBC: White blood cell count. Hematopoietic populations were analyzed in flow cytometry and colony formation in methylcellulose assay. Data are expressed as mean ± standard deviation, analyzed by Student t test and considered statistically significant (*) if p ≤ 0.05. DisclosuresNo relevant conflicts of interest to declare.

CXCL12/CXCR4 signaling counteracts docetaxel-induced microtubule stabilization via p21-activated kinase 4-dependent activation of LIM domain kinase 1.

Emerging data highlight the significance of chemokine (C-X-C motif) ligand 12/chemokine (C-X-C motif) receptor 4 (CXCL12/CXCR4) signaling axis in the chemoresistance of several malignancies, including prostate cancer (PCa); however, underlying mechanisms remain largely elusive. Here, we demonstrate that CXCL12 treatment rescues the PCa cells from docetaxel (DTX)-induced toxicity by overriding its effect on cell cycle (G2/M phase arrest). We further demonstrate that the chemoprotective effect of CXCL12 is abolished upon pharmacological inhibition or RNA interference-mediated silencing of CXCR4. Moreover, microtubule stabilization caused by DTX is suppressed in CXCL12-stimulated PCa cells as revealed by immunofluorescence and immunoblot analyses. The effect of CXCL12 on microtubule stabilization is abrogated when PCa cells are pre-treated with a CXCR4 antagonist. In additional studies, we show that the chemoprotective action of CXCL12/CXCR4 signaling is mediated by p21-activated kinase 4 (PAK4)-dependent activation of Lim domain kinase 1 (LIMK1), and inhibition of either PAK4 or LIMK1 leads to re-sensitization of PCa cells to DTX-induced tubulin polymerization and cellular toxicity even in the presence of CXCL12. Altogether, our findings uncover a novel mechanism underlying CXCL12/CXCR4 signaling-induced PCa chemoresistance and suggest that targeting of this signaling axis or its downstream effector pathway could lead to therapeutic enhancement of DTX.

CXCR4 expression affects overall survival of HCC patients whereas CXCR7 expression does not.

Hepatocellular carcinoma (HCC) is a heterogeneous disease with a poor prognosis and limited markers for predicting patient survival. Because chemokines and chemokine receptors play numerous and integral roles in HCC disease progression, the CXCR4–CXCL12–CXCR7 axis was studied in HCC patients. CXCR4 and CXCR7 expression was analyzed by immunohistochemistry in 86 HCC patients (training cohort) and validated in 42 unrelated HCC patients (validation cohort). CXCR4 levels were low in 22.1% of patients, intermediate in 30.2%, and high in 47.7%, whereas CXCR7 levels were low in 9.3% of patients, intermediate in 44.2% and high in 46.5% of the patients in the training cohort. When correlated to patient outcome, only CXCR4 affected overall survival (P=0.03). CXCR4–CXCL12–CXCR7 mRNA levels were examined in 33/86 patients. Interestingly, the common CXCR4–CXCR7 ligand CXCL12 was expressed at significantly lower levels in tumor tissues compared to adjacent normal liver (P=0.032). The expression and function of CXCR4 and CXCR7 was also analyzed in several human HCC cell lines. CXCR4 was expressed in Huh7, Hep3B, SNU398, SNU449 and SNU475 cells, whereas CXCR7 was expressed in HepG2, Huh7, SNU449 and SNU475 cells. Huh7, SNU449 and SNU475 cells migrated toward CXCL12, and this migration was inhibited by AMD3100/anti-CXCR4 and by CCX771/anti-CXCR7. Moreover, SNU449 and Huh7 cells exhibited matrix invasion in the presence of CXCL12 and CXCL11, a ligand exclusive to CXCR7. In conclusion, CXCR4 affects the prognosis of HCC patients but CXCR7 does not. Therefore, the CXCR4–CXCL12–CXCR7 axis plays a role in the interaction of HCC with the surrounding normal tissue and represents a suitable therapeutic target.

CXCR4 and CXCR7 transduce through mTOR in human renal cancer cells.

Treatment of metastatic renal cell carcinoma (mRCC) has improved significantly with the advent of agents targeting the mTOR pathway, such as temsirolimus and everolimus. However, their efficacy is thought to be limited by feedback loops and crosstalk with other pathways leading to the development of drug resistance. As CXCR4–CXCL12–CXCR7 axis has been described to have a crucial role in renal cancer; the crosstalk between the mTOR pathway and the CXCR4–CXCL12–CXCR7 chemokine receptor axis has been investigated in human renal cancer cells. In SN12C and A498, the common CXCR4–CXCR7 ligand, CXCL12, and the exclusive CXCR7 ligand, CXCL11, activated mTOR through P70S6K and 4EBP1 targets. The mTOR activation was specifically inhibited by CXCR4 antagonists (AMD3100, anti-CXCR4-12G5 and Peptide R, a newly developed CXCR4 antagonist) and CXCR7 antagonists (anti-CXCR7-12G8 and CCX771, CXCR7 inhibitor). To investigate the functional role of CXCR4, CXCR7 and mTOR in human renal cancer cells, both migration and wound healing were evaluated. SN12C and A498 cells migrated toward CXCL12 and CXCL11; CXCR4 and CXCR7 inhibitors impaired migration and treatment with mTOR inhibitor, RAD001, further inhibited it. Moreover, CXCL12 and CXCL11 induced wound healing while was impaired by AMD3100, the anti CXCR7 and RAD001. In SN12C and A498 cells, CXCL12 and CXCL11 promoted actin reorganization characterized by thin spikes at the cell periphery, whereas AMD3100 and anti-CXCR7 impaired CXCL12/CXCL11-induced actin polymerization, and RAD001 treatment further reduced it. In addition, when cell growth was evaluated in the presence of CXCL12, CXCL11 and mTOR inhibitors, an additive effect was demonstrated with the CXCR4, CXCR7 antagonists and RAD001. RAD001-resistant SN12C and A498 cells recovered RAD001 sensitivity in the presence of CXCR4 and CXCR7 antagonists. In conclusion, the entire axis CXCR4–CXCL12–CXCR7 regulates mTOR signaling in renal cancer cells offering new therapeutic opportunities and targets to overcome resistance to mTOR inhibitors.

Opposing effects of high- and low-molecular weight hyaluronan on CXCL12-induced CXCR4 signaling depend on CD44

The tumor microenvironment makes a decisive contribution to the development and dissemination of cancer, for example, through extracellular matrix components such as hyaluronan (HA), and through chemokines that regulate tumor cell behavior and angiogenesis. Here we report a molecular link between HA, its receptor CD44 and the chemokine CXCL12 in the regulation of cell motility and angiogenesis. High-molecular-weight HA (hHA) was found to augment CXCL12-induced CXCR4 signaling in both HepG2iso cells and primary human umbilical vein endothelial cells, as evidenced by enhanced ERK phosphorylation and increased cell motility. The augmentation of CXCR4 signaling translated into increased vessel sprouting and angiogenesis in a variety of assays. Small HA oligosaccharides (sHA) efficiently inhibited these effects. Both siRNA-mediated reduction of CD44 expression and antibodies that block the interaction of CD44 with HA provided evidence that CXCL12-induced CXCR4 signaling depends on the binding of hHA to CD44. Consistently, CD44 and CXCR4 were found to physically interact in the presence of CXCL12, an interaction that could be inhibited by sHA. These findings provide novel insights into how microenvironmental components interact with cell surface receptors in multi-component complexes to regulate key aspects of tumor growth and progression.

Expression of the chemokines CXCL12 and CX3CL1 and their receptors in human nerve sheath tumors.

Peripheral nerve sheath tumors are in most cases slowly growing neoplasms that can be adequately cured by surgical resection. However, facing the risk of a neurosurgical intervention and the trend of multiple relapses of nerve sheath tumors the development of additional therapy strategies seems to be favourable, and therefore substantiated knowledge of molecular and cellular mechanisms in nerve sheath tumors should be achieved. Here, we firstly describe the expression of the chemokines CXCL12 (SDF-1) and CX3CL1 (fractalkine) and their respective receptors CXCR4, CXCR7 and CX3CR1 in different entities of human nerve sheath tumors and normal control tissues. Both ligands and their receptors are expressed in high to moderate levels on mRNA and protein level in benign and malignant nerve sheath tumors. While CXCL12 was mainly found in schwannoma cells (S100⁺) in situ, its receptor CXCR4 is also partly found on CD11b-positive macrophages / microglia and its alternative receptor CXCR7 is also expressed by endothelial cells and macrophages. CX3CL1 is expressed by parts of the schwannoma and endothelial cells, whereas its receptor CX3CR1 is expressed by nearly all tumor cells and macrophages, but not by endothelial cells. Taken together, we could show the presence of CXCL12 and CX3CL1 and their respective receptors in benign and malignant human nerve sheath tumors. Further investigations may show their functional role in health and disease.

Cortactin Expression Is Tightly Connected to B-Cell Chronic Lymphocytic Leukemia Aggressiveness

AbstractAbstract 4561 Background.Cortactin is an ubiquitous actin-binding protein, encoded by EMS1 gene and localized in chromosome 11q13 region. This protein is expressed in nearly all mammalian tissues and mostly localizes to dynamic actin structures. As a consequence, cortactin is involved in regulating several actin-dependent processes, including lamellipodium protrusion, trafficking of the key invadopodia proteases and intracellular transducer downstream to kinase-mediated cell signalling upon phosphorylation by Src and Syk tyrosine kinase families. Cortactin is over-expressed in several tumors, such as esophageal squamous cell carcinoma, head and neck squamous cell carcinoma and gastric carcinoma, and so tied to tumor aggressiveness by the promotion of cell invasion and metastasis. We previously showed that cortactin is over-expressed also in neoplastic B cells of patients with B-Cell Chronic Lymphocytic Leukemia (B-CLL). In the present study we investigated the regulation of cortactin activity by the Src kinase Lyn in neoplastic B-CLL cells and the biological implications of cortactin overexpression in this leukemia. Methods.Twenty patients and ten normal controls were enrolled. Informed consent was obtained from all patients according to the Declaration of Helsinki. CD19+ lymphocytes were purified from peripheral blood by negative selection using the RosetteSep cells isolation kit (StemCell Technologies). Cortactin localization was performed by Confocal Microscope analysis at basal condition, in presence of the chemotactic stimulus CXCL12 and Src kinase inhibitor PP2. Lyn kinase and cortactin phosphorylation levels were evaluated by western blotting analysis. Migration assay was performed using 3 μm pore filters (Transwell Permeable Supports) in the presence of CXCL12, Src kinase inhibitor PP2 and after cortactin silencing. Results.We found that cortactin localization is regulated by Lyn kinase through its phosphorylation. In CLL cells, cortactin was over-phosphorylated and it did not co-localize with actin, as compared to normal cells. PP2-inhibition of Lyn decreased cortactin phosphorylation and triggered its co-localization with actin. Cortactin over-expression proved to be associated to the increased B-CLL spreading through Lyn activity. In fact, not only cortactin over-expression correlated with leukemic cell increased response to CXCL12 (r = 0.9), but also the inhibition of cortactin activity, through PP2 inhibitor or cortactin silencing, drastically reduced neoplastic cell migration after chemotactic triggering. Conclusions.These results suggest that cortactin is involved in aggressiveness and spreading of B-CLL cells and that Lyn-cortactin axis could represent an alternative target for the development of new therapeutic strategies. Disclosures:No relevant conflicts of interest to declare.

Multiple Myeloma Cells Adhere to Netrin-1 Via Heparin-Sulphate Moieties

Abstract 3952A major obstacle to the treatment of Multiple Myeloma (MM) is the localization of myeloma cells to the bone marrowstroma, enabling drug resistance. The exact mechanisms of adhesion of myeloma cells to the bone marrow are not known, but adhesion molecules and chemokine signals, in particular vascular cell adhesion protein 1 (VCAM-1) and C-X-C chemokine 12 (CXCL12) which control bone marrow tropism, are thought to be the main players. Netrin-1, which acts as an axonal guidance cue, plays a role in leukocyte migration in lymph nodes and in atherosclerotic lesions, but has not been tested as a substrate for myeloma cell adhesion previously. Based on expression of the netrin-1 receptor Deleted in Colorectal Cancer (DCC) on activated human B cells, we tested the ability of myeloma cells to adhere to netrin-1, an axonal guidance cue. Using interference reflection microscopy (IRM) which employs the method of interference of light reflected from nearby surfaces to measure cell-substratum distances and cell-substratum adhesion, we assessed cell adhesion and cell spreading on substrates immobilized on glass. Here, we used this technique to assess myeloma cell adhesion and migration on various substrates and found netrin-1 to be an exceptional adhesion ligand for myeloma cells.We prepared glass substrates coated with the recombinants ligands intercellular adhesion molecule,ICAM-1(50μM), and VCAM-1(50μM), with or without chemokine ligand, CXCL12(0.1mg/mL), which have been implicated in plasma cells and myeloma cell migration, previously. We used freshly purified, fluorescently-labeled primary myeloma cells from newly diagnosed patients, prior to any treatment. Using IRM, we imaged the cell contacting the substrate in order to measure adhesion and differentiate crawling versus fluid flow movement. Based on the IRM image, we could calculate the fraction of cells in the field that were adhered to the substrate, and compared between conditions and for various patient samples. We observed that myeloma cells can adhere and migrate slowly on VCAM-1 in the presence of CXCL12, but are unable to adhere to ICAM-1 with or without chemokines. We tested myeloma cell binding to netrin-1 and saw a strong adhesion 60–90% of cells in 7 out of 9 patients samples tested. The cell spreading on netrin-1 was more than 3 times larger than on VCAM-1 with CXCL12 substrates. Expression of netrin-1 in the bone marrow has not been determined yet nor its role in MM. Heparin-like molecule, SST0001, has been tested in myeloma studies, in an attempt to interfere with heparinase activity and syndecan-1 shedding, and tumor growth. We tested pre-blocking netrin-1 substrates with heparin and observed elimination of greater than 95% of myeloma cell adhesion in all patients samples tested. Treating patients with heparin-like molecules may have additional functions, by blocking binding to netrin-1 and soluble signals that contain heparin-binding domains. Reciprocally, blocking heparin-sulfated groups with netrin-1 may block myeloma cell adhesion and can be used to targeting strategy for chemotherapeutic drugs as well. Disclosures:No relevant conflicts of interest to declare.

In vitro and in vivo therapeutic efficacy of CXCR4 antagonist BKT140 against human non–small cell lung cancer

CXCR4/CXCL12 interactions promote non-small cell lung cancer (NSCLC) growth and dissemination. Furthermore, this axis might promote NSCLC resistance to chemotherapy and/or radiotherapy. Therefore, the CXCR4/CXCL12 axis constitutes an attractive therapeutic target for the treatment of NSCLC. We aimed to characterize the therapeutic efficacy of the novel CXCR4 antagonist BKT140 against human NSCLC. We determined the CXCR4 expression in 5 NSCLC cell lines (H358, A549, H460, H1299, and L4). We then tested the colony-forming capacity and proliferation of these cells in the presence of CXCL12 and BKT140. Next, we measured the in vivo growth of A549 and H460 xenografts with or without BKT140 treatment. Finally, we examined, in vitro, the potential antiproliferative effect of BKT140 combined with cisplatin or paclitaxel and after irradiation of NSCLC cells. All tested cell lines expressed CXCR4 and showed increased colony formation in response to CXCL12 stimulation. BKT140 reduced the colony-forming capacity of NSCLC cells. Proliferation assays demonstrated both cytotoxic and cytostatic properties for this peptide. H460 cells were the most sensitive to BKT140 and A549 cells the least. Subcutaneous administration of BKT140 significantly delayed the development of H460 xenografts and showed a similar trend for A549 xenografts. Finally, the antiproliferative effects of BKT140 appears to be additive to those of chemotherapeutic drugs and radiotherapy. Targeting the CXCL12/CXCR4 axis with BKT140 attenuated NSCLC cells tumor growth and augmented the effects of chemotherapy and radiotherapy. Future research will benefit from delineating the downstream mechanism of BKT140 action and defining BKT140 susceptibility markers.

Similarities and Differences in Signal Transduction and Crosstalk of the G Protein-Coupled Receptors CXCR4 and CysLT1 in CD34+ Hematopoietic Stem/Progenitor Cells,

Abstract 3392CD34+ hematopoietic stem- and progenitor cells (HPC) express high levels of the G protein-coupled receptors (GPCR) CXCR4 and CysLT1. In contrast to the established role of CXCR4 in stem cell homing, the function of CysLT1 in HPC remains only partially understood. We found that upon stimulation of peripheral blood CD34+ HPC in vitro with the respective ligands (CXCL12/SDF1 for CXCR4 and the cysteinyl-leukotriene LTD4 for CysLT1), both receptors similarly mediate the following functional activities: intracellular calcium fluxes, actin polymerization, adhesion to endothelium in vitro and chemotaxis. By Westernblot analysis, we could demonstrate Pyk2- and MAP-kinase phosphorylation as pivotal elements of CysLT1 signaling. These pathways have previously been identified also in CXCR4 signaling.To further analyze signal transduction pathways of both receptors, CD34+ cells were pretreated with pertussis toxin (PTX) or with a specific PKC inhibitor, which is an isoform-specific inhibitory myristoylated peptide derived from the pseudosubstrate (PS) region of PKCzeta, mimics the substrate, and maintains PKC in its nonactive isoform. Subsequently, we determined actin polymerization by flow cytometry using phalloidin-FITC, and adhesion to IL-1 stimulated endothelial cells (HUVEC). CXCR4 signaling leading to actin polymerization was found to be completely blocked by preincubation with pertussis toxin (PTX) and therefore is mediated exclusively by Gi proteins, while CysLT1 also involves Gq proteins as reflected by only partial inhibition by PTX. For both receptors, the Pyk2 signaling pathway leading to actin polymerization and adhesion was completely suppressed by preincubation with PSzeta and therefore dependent on atypical PKCzeta, which is calcium and DAG independent.We further examined whether these two similarly functioning receptors maintain any crosstalk, as has been reported for GPCR. Their possible interaction was explored using actin polymerization as a functional read-out. CXCR4- and CysLT1-mediated actin polymerization in response to their respective ligand was induced within 10 sec and returned to basal levels after 4 min. A second challenge after 4 min with the same ligand resulted in a complete suppression, demonstrating self-desensitization of both receptors. Interestingly, restimulating CXCL12-induced cells with LTD4 after 4 min resulted in complete suppression of actin polymerization, whereas restimulating LTD4-induced cells with CXCL12 lead to F-actin levels comparable to those achieved with the first challenge. These data show for the first time that CXCR4 can cross-desensitize CysLT1 while there is no crosstalk from CysLT1 to CXCR4.In conclusion, CXCR4 and CysLT1 share major signaling pathways. However, the ability to desensitize other GPCR is strikingly different. The finding that CXCR4 cross-desensitizes CysLT1 but not vice versa could explain our observation that a CysLT1 antagonist (montelukast) did not mobilize HPC in vivo, as the presence of CXCL12 in the stem cell niche may result in desensitization of CysLT1. In contrast, CXCR4-dependent bone marrow homing may not be influenced by conditions with high local and systemic cysteinyl-leukotriene concentrations, e.g. during allergy and inflammation. In the absence of CXCR4 activation however, CysLT1 could be important for homing of stem and progenitor cells in areas other than the bone marrow with a high local concentration of cysteinyl leukotrienes, e.g. in inflamed tissues. Disclosures:No relevant conflicts of interest to declare.