CXCR4 Blockade Research Articles

A novel CXCR4 antagonist counteracts paradoxical generation of cisplatin-induced pro-metastatic niches in lung cancer

Platinum-based chemotherapy remains widely used in advanced non-small cell lung cancer (NSCLC) despite experimental evidence of its potential to induce long-term detrimental effects, including the promotion of pro-metastatic microenvironments. In this study, we investigated the interconnected pathways underlying the promotion of cisplatin-induced metastases.In tumor-free mice, cisplatin treatment resulted in an expansion in the bone marrow of CCR2+CXCR4+Ly6Chigh inflammatory monocytes (IMs) and an increase in lung levels of stromal SDF-1, the CXCR4 ligand. In experimental lung metastasis assays, cisplatin-induced IMs promoted the extravasation of tumor cells and the expansion of CD133+CXCR4+ metastasis-initiating cells (MICs). Peptide R, a novel CXCR4 inhibitor designed as an SDF-1 mimetic peptide, prevented cisplatin-induced IM expansion, the recruitment of IMs into the lungs, and the promotion of metastasis. At the primary tumor site, cisplatin treatment reduced tumor size while simultaneously inducing tumor release of SDF-1, MIC expansion, and recruitment of pro-invasive CXCR4+ macrophages. Co-recruitment of MICs and CCR2+CXCR4+ IMs to distant SDF-1-enriched sites also promoted spontaneous metastases that were prevented by CXCR4 blockade. In clinical specimens from NSCLC patients SDF-1 levels were found to be higher in platinum-treated samples and related to a worse clinical outcome. Our findings reveal that activation of the CXCR4/SDF-1 axis specifically mediates the pro-metastatic effects of cisplatin and suggest CXCR4 blockade as a possible novel combination strategy to control metastatic disease.

Tenascin‐C immobilizes infiltrating T lymphocytes through CXCL12 promoting breast cancer progression

Immune checkpoint therapy, where CD8 tumor infiltrating T lymphocytes (TIL) are reactivated, is a promising anti‐cancer treatment approach, yet with low response rates. The extracellular matrix, in particular tenascin‐C, may generate barriers for TIL. To investigate this possibility, we used a MMTV‐NeuNT and syngeneic mammary gland grafting model derived thereof with engineered tenascin‐C levels and observed accumulation of CD8 TIL in tenascin‐C‐rich stroma. Inhibition studies revealed that tenascin‐C induced CXCL12 through TLR4. By binding CXCL12, tenascin‐C retained CD8 TIL in the stroma. Blockade of CXCR4, the receptor of CXCL12, enhanced macrophage and CD8 TIL infiltration and reduced tumor growth and subsequent metastasis. Retention of CD8 TIL by tenascin‐C/CXCL12 was also observed in human breast cancer by tissue staining. Moreover, whereas high CD8 TIL numbers correlated with longer metastasis‐free survival, this was not the case when also tenascin‐C and CXCL12 levels were high. Altogether, these results may be useful for improving tumor immunity as diagnostic tool and to formulate a future “TIL‐matrix‐release‐and‐reactivate” strategy.

Improving anti-PD-L1 therapy in triple negative breast cancer by polymer-enhanced immunogenic cell death and CXCR4 blockade

Triple negative breast cancer (TNBC) with highly metastatic features generally does not respond to anti–programmed cell death 1 ligand 1 (PD-L1) therapy due to multiple immunosuppressive mechanisms to exclude and disable T cells. Here, we develop a polymer-based combinatory approach consisting of both immunogenic cell death (ICD)-inducing and CXCR4-inhibiting function to prime tumor microenvironment and improve anti-PD-L1 therapy in TNBC. Our findings revealed that the combination therapy was able to spur the T cell response in primary tumors by increasing the tumor immunogenicity to recruit T cells, removing the physiological barriers of intratumoral fibrosis and collagen to increase T cell infiltration, and reducing the immunosuppressive cells to revive T cells. Meanwhile, such approach efficiently inhibited the formation of pre-metastatic niche in abscopal lung. Because of the significant promotion of anti-tumor and anti-metastasis immunity, the non-responding TNBC gained robust responsiveness to anti-PD-L1 therapy which resulted in complete eradication of orthotopic tumors, inhibition of pulmonary metastasis, and durable memory effects against tumor recurrence. Our work provided a generalizable approach of simultaneous ICD induction and CXCR4 blockade to apply anti-PD-L1 therapy in TNBC.

CXCR4 signaling controls dendritic cell location and activation at steady state and in inflammation

AbstractDendritic cells (DCs) encompass several cell subsets that collaborate to initiate and regulate immune responses. Proper DC localization determines their function and requires the tightly controlled action of chemokine receptors. All DC subsets express CXCR4, but the genuine contribution of this receptor to their biology has been overlooked. We addressed this question using natural CXCR4 mutants resistant to CXCL12-induced desensitization and harboring a gain of function that cause the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS), a rare immunodeficiency associated with high susceptibility to the pathogenesis of human papillomavirus (HPV). We report a reduction in the number of circulating plasmacytoid DCs (pDCs) in WHIM patients, whereas that of conventional DCs is preserved. This pattern was reproduced in an original mouse model of WS, enabling us to show that the circulating pDC defect can be corrected upon CXCR4 blockade and that pDC differentiation and function are preserved, despite CXCR4 dysfunction. We further identified proper CXCR4 signaling as a critical checkpoint for Langerhans cell and DC migration from the skin to lymph nodes, with corollary alterations of their activation state and tissue inflammation in a model of HPV-induced dysplasia. Beyond providing new hypotheses to explain the susceptibility of WHIM patients to HPV pathogenesis, this study shows that proper CXCR4 signaling establishes a migration threshold that controls DC egress from CXCL12-containing environments and highlights the critical and subset-specific contribution of CXCR4 signal termination to DC biology.

Glucocorticoid-induced eosinopenia results from CXCR4-dependent bone marrow migration

AbstractGlucocorticoids are considered first-line therapy in a variety of eosinophilic disorders. They lead to a transient, profound decrease in circulating human eosinophils within hours of administration. The phenomenon of glucocorticoid-induced eosinopenia has been the basis for the use of glucocorticoids in eosinophilic disorders, and it has intrigued clinicians for 7 decades, yet its mechanism remains unexplained. To investigate, we first studied the response of circulating eosinophils to in vivo glucocorticoid administration in 3 species and found that the response in rhesus macaques, but not in mice, closely resembled that in humans. We then developed an isolation technique to purify rhesus macaque eosinophils from peripheral blood and performed live tracking of zirconium-89-oxine–labeled eosinophils by serial positron emission tomography/computed tomography imaging, before and after administration of glucocorticoids. Glucocorticoids induced rapid bone marrow homing of eosinophils. The kinetics of glucocorticoid-induced eosinopenia and bone marrow migration were consistent with those of the induction of the glucocorticoid-responsive chemokine receptor CXCR4, and selective blockade of CXCR4 reduced or eliminated the early glucocorticoid-induced reduction in blood eosinophils. Our results indicate that glucocorticoid-induced eosinopenia results from CXCR4-dependent migration of eosinophils to the bone marrow. These findings provide insight into the mechanism of action of glucocorticoids in eosinophilic disorders, with implications for the study of glucocorticoid resistance and the development of more targeted therapies. The human study was registered at ClinicalTrials.gov as #NCT02798523.

Bone Marrow (BM) Delivery of Genetically-Modified (gm) Adult CD34+ Hematopoietic Stem and Progenitor Cells (HSPC) Improves Homing and Engraftment of Short-Term Progenitors over Long-Term Repopulating Hematopoietic Stem Cells

Gene therapy/editing of CD34+ HSPC ex vivo, followed by their transplantation, can cure a variety of hematologic diseases. However, a substantial loss of HSPC occurs from collection to transplant. Losses occur during processing for HSPC enrichment, ex vivo genetic manipulation and culture, formulation, and testing prior to transplant. Further, HSPC are lost to peripheral organs during homing when delivered intravenously (IV), reducing the effective gm HSPC dose; a loss compounded by the lack of helper cells that aid in the homing and engraftment process which are removed during enrichment. Direct BM delivery of gm HSPC can overcome some of these limitations. This has been tried previously, with non-enriched whole cord blood (CB) and non-gm HSPC, with conflicting results. We hypothesized that BM delivery of a limited dose of gm adult HSPC would improve long-term repopulation over that of IV delivery by bypassing HSPC loss during homing. Using bioluminescent imaging, we determined that CB HSPC transduced with a luciferase lentiviral vector (LV) delivered by intra-femoral (IF) injection localized to the injected femur, validating our injection method. Next, we delivered mobilized peripheral blood (MPB) HSPC transduced with a GFP LV into irradiated NOD.LtSz-scid IL2rg -/- (NSG) mice via IV or IF injection in limiting dilution. Total human engraftment (hCD45+ cells), transduced human engraftment (hCD45+GFP+ cells), and multi-lineage engraftment were measured in the BM at 3- and 6-months post-transplant. HSPC gave rise to a bi-lineage (B-myeloid) graft at 3 months, suggesting hematopoietic progenitor cell (HPC) engraftment, and a multi-lineage graft (hCD33+, hCD19+, hCD3+, and hCD34+ cells) at 6 months, suggesting engraftment from a long-term repopulating cell or hematopoietic stem cell (HSC). At 3 months, IF delivery of HSPC resulted in significantly higher total and transduced human cell engraftment, measured in the non-injected femur (Table 1). The engraftment was bi-lineage. At 6 months, IF delivery of HSPC no longer significantly increased engraftment over IV delivery (Table 1). However, a multi-lineage graft was present, indicating full hematopoietic repopulation. There was no significant difference in the lineage output between either delivery method at 3 or 6 months. These data suggest that HPC homed and engrafted more efficiently than HSC, when delivered IF. Alternatively, IF delivery altered the BM microenvironment, allowing preferential homing of HPC. However, CD34- cells injected IF, to simulate pressure and passage of cells through the BM with IF delivery, followed by IV delivery of CD34+ cells (sham IF with IV HSPC delivery) resulted in similar homing patterns to CD34+ cells delivered IV (p=0.1, Figure 1A), suggesting that differences between IV and IF delivery were likely due to cell-intrinsic rather than cell-extrinsic differences between HPC and HSC. To study the mechanism of preferential engraftment of HPC over HSC with IF delivery, we analyzed expression of the major homing receptors CXCR4 and VLA-4 on HPC and HSC. CXCR4 (Figure 1B) and VLA-4 were both expressed at significantly higher levels on HPC than on HSC (CXCR4 p<0.01; VLA-4 p<0.05) and their expression increased with increasing culture time and with HSPC cycling. However, VLA-4 expression was significantly increased in GFP+ (MFI 65313 ± 4750) compared to GFP- (MFI 48969 ± 2099; p<0.01) HSPC. CXCR4 expression was similar in both GFP+ (MFI 4261 ± 189) and GFP- (MFI 5245 ± 1186) HSPC, mimicking the in vivo engraftment pattern of GFP+ and GFP- cells, suggesting that CXCR4 may be the molecule responsible for enhancing HPC homing and engraftment with BM delivery. An initial experiment shows that when we remove the high CXCR4 expressing CD34+38+ HPC and deliver HSC-enriched CD34+38- cells IV or IF, IF delivery results in higher long-term engraftment (additional experiments ongoing, Figure 1C, D). These data support the hypothesis that cell-intrinsic differences in the homing behavior of HSC and HPC is likely due to their differential expression of CXCR4. Studies underway on blockade of CXCR4 or VLA-4 on gm HPC and/or gm HSC followed by their IF or IV delivery will be presented. Overall, we show IV delivery of gm HSPC is comparable to BM delivery. However, as HSC-enriched cells become clinically available for genetic therapies, BM delivery of enriched gm HSC may result in superior engraftment. Disclosures Malik: Aruvant Sciences, Forma Therapeutics, Inc.: Consultancy; Aruvant Sciences, CSL Behring: Patents & Royalties.

Combined Inhibition of CXCR4 Signaling and System xc- Transporter Activity Induces Synthetic Lethality in T-ALL Cells By Suppressing Gsh and Inducing Ferroptosis

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 10-15% of pediatric and 25% of adult ALL cases. Prior studies have established that most cases pf T-ALL are addicted to CXCR4 signaling. Indeed, strong preclinical data demonstrating therapeutic activity of BL-8040, a potent CXCR4 antagonist, have led to a clinical trial of BL-8040 in combination with nelarabine for patients with relapsed/refractory T-ALL (NCT02763384). However, the molecular mechanisms by which CXCR4 blockade induces T-ALL cell death are unknown. Using a human T-ALL xenotransplantation model, we previously reported that treatment with BL-8040 killed T-ALL cells through a non-apoptotic mechanism. Transcriptome sequencing revealed that BL-8040 induced alterations in genes involved in oxidative phosphorylation and carbohydrate metabolism. Indeed, seahorse experiments show that BL-8040 markedly reduced both oxidative phosphorylation and glycolysis. However, metabolic tracing studies using 13C-labeled glucose show that BL-8040 treatment does not have a major effect on the contribution of glucose to either glycolysis or the citric acid cycle. Instead, the major alteration observed is the reduced entry of glucose into the pentose phosphate pathway (PPP). A major function of the PPP pathway is to generate NADPH, which regulates reactive oxygen species (ROS) by producing reduced glutathione (GSH). Indeed, BL-8040 treatment resulted in a significant decrease in the ratio of reduced glutathione to oxidized glutathione. Together, these data suggest that BL-8040 induces oxidative stress by inhibiting GSH production. One mechanism utilized by cancer cells to regulate GSH levels and oxidative stress is the system xc- amino acid antiporter that mediates the exchange of extracellular L-cystine and intracellular l-glutamate across the plasma membrane, resulting in the production of GSH and oxidative protection. We measured L-cystine levels in the media of T-ALL cells cultured for 24 hours with or without BL-8040. A significant decrease in L-cystine in the media was observed. These data, along with increased expression of the xc- transporter (SLC7A11), suggested that increased system xc- activity was compensating for the loss of GSH induced by BL-8040. To test this possibility, we cultured T-ALL cells in L-cystine deficient media. Loss of L-cystine in the media resulted in a modest decrease in T-ALL cell viability that was markedly increased, in a synergistic fashion, upon treatment with BL-8040. Interestingly, caspase 3 was not activated, suggesting that, similar to in vivo results, BL-8040 induces a non-apoptotic cell death. This observation, coupled with the reduction in GSH, suggested the hypothesis that BL-8040 induces ferroptosis. Consistent with the hypothesis, treatment of T-ALL cells with ACXT-3102, a novel system xc- inhibitor, significantly enhanced BL-8040 killing of T-ALL cells in vitro. Collectively, these data suggest that T-ALL cells are sensitive to perturbations of the glutathione axis. Combined inhibition of CXCR4 signaling and system xc- activity exploits this vulnerability and presents a promising new therapeutic approach for T-ALL. Disclosures Uy: Astellas Pharma: Honoraria; Jazz Pharmaceuticals: Consultancy; Genentech: Consultancy; Agios: Consultancy; Pfizer: Consultancy; Daiichi Sankyo: Consultancy. Sorani:BiolineRx Ltd: Current Employment. Vainstein:BiolineRx Ltd: Current Employment. Davish:BiolineRx Ltd: Current Employment. Hawkins:Accuronix Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Abstract 1106: Cisplatin-induced activation of SDF-1/CXCR4 axis sustains lung cancer metastasis by promoting co-recruitment of metastasis initiating cells and inflammatory monocytes

Abstract Background: Platinum-based treatment of locally advanced non-small cell lung cancer (NSCLC) provides some clinical benefit in controlling local disease but is ineffective on metastatic dissemination. Rising evidence suggests that chemotherapy can promote an inflammatory reaction supportive of metastasis growth.We previously demonstrated that cisplatin treatment of mouse xenografts enriches for the chemoresistant fraction of CD133+CXCR4+ lung cancer metastasis initiating cells (MICs), increasing distant metastasis development. We hypothesize here that the SDF-1/CXCR4 axis, implied in MICs migration and in stromal cells trafficking, could play a critical role in cisplatin-induced pro-metastatic effects. Results: To study the effects of cisplatin in promoting a pre-metastatic niche, tumor-free naïve SCID mice were treated with cisplatin plus/minus peptide R, a novel inhibitor of CXCR4. Cisplatin resulted in a rapid expansion of CCR2+CXCR4+Ly6Chigh inflammatory monocytes (IM) in the bone marrow, concomitantly with their recruitment to murine lungs guided by enhanced endothelial release of SFD-1. Tail-vein injection of H460 human lung cancer cells 72h after cisplatin administration resulted in augmented number of lung metastases enriched in CD133+CXCR4+ MICs. We proposed that the abundance of CXCR4+IM together with increased endothelial permeability caused by cisplatin favors tumor cells extravasations and the expansion of MICs through SDF-1/CXCR4 axis activation. Combination treatment with a CXCR4 inhibitor prevented IM recruitment, MICs expansion and consequently abolished metastasis overgrowth induced by cisplatin. Combination treatment of H460 subcutaneous xenografts in a regimen mimicking clinical setting revealed that cisplatin also caused tumor release of SDF-1 able to trigger local expansion of MICs subset and to recruit CXCR4+tumor associated macrophages involved in tumor cells intravasation. Stromal SDF-1 induced by cisplatin at distant site also co-recruited MICs and CCR2+CXCR4+IM, promoting spontaneous metastasis formation that can be counteracted by CXCR4 blockade. We confirmed in clinical setting that platinum-based neoadjuvant treatment of NSCLC patients significantly increases tumor SDF-1 expression. Moreover, an higher expression of tumor SDF-1 after cisplatin neo-adjuvant treatment was associated with patients' shorter DFS (p=0,0056; Hazards Ratio= 3,1) and poor OS (p=0,029; Hazards Ratio= 3,46). Conclusions: Our data reveal a paradoxical pro-metastatic effect of cisplatin that fosters MIC-IM recruitment and cross-talk via SDF-1/CXCR4 axis activation. A new combination strategy based on CXCR4 inhibition may disrupt these interactions, providing more effective results for lung cancer treatment Citation Format: Giulia Bertolini, Valeria Cancila, Orazio Fortunato, Giuseppe Lo Russo, Monica Tortoreto, Giovanni Centonze, Massimo Milione, Claudio Tripodo, Stefania Scala, Gabriella Sozzi, Luca Roz. Cisplatin-induced activation of SDF-1/CXCR4 axis sustains lung cancer metastasis by promoting co-recruitment of metastasis initiating cells and inflammatory monocytes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1106.

Abstract 6325: CXCR4 intracellular protein regulates chemotherapy resistance by modulating the expression of death receptor 5

Abstract Chemotherapy resistance is one of the pivotal reasons for cancer-associated huge mortality and morbidity. Cancer cells become therapy resistant by smartly evading the execution of apoptosis. Death Receptors (DR) are critical pro-apoptotic factors that regulate cell death, and paucity of death receptors in cancer cells promotes therapy resistance. High expression of CXCR4 is strongly correlated with poor prognosis and adverse clinical outcomes. Inhibitors of the CXCL12-CXCR4 axis, such as FDA approved drug plerixafor (AMD3100), and Nox-A12 have shown limited clinical success in cancer treatment. Alternately, CXCR7, another CXCL12 binding receptor, could be the reason for the limited success of the CXCR4 blockade in clinics. However, recent preclinical data for blocking both the chemokine receptors failed to explain the pro-tumorigenic functions of these axes. Therefore, CXCR4-CXCL12 signaling may not be the exclusive contributor to its pro-tumorigenic functions. In our continuous effort to understand the chemokine receptor signaling inhibition as cancer therapy, here we unexpectedly discovered that instead of CXCR4-CXCL12 signaling, CXCR4 intracellular protein augments therapy resistance and pro-tumorigenic functions. By utilizing stable gain and loss of function approaches, we observed that CXCR4 selectively resists and sensitizes cancer cells against paclitaxel therapy. Our unbiased apoptosis array followed by immunoblot, FACS, real-time PCR, and ChIP analysis clearly demonstrate that CXCR4 promotes downregulation of pro-apoptotic protein DR5 via modulating differential recruitment of transcription factors p53 and YY1 to the promoter of DR5 gene. Inhibiting CXCR4 mediated signals failed to block the above phenotype. Irrespective of CXCR4 surface expression, three different colon cancer xenograft models evidently exhibited loss of CXCR4 protein results in compromised colon tumor growth in vivo. Finally, performing TCGA data mining, real-time PCR, immunofluorescence microscopy of human cancer patient samples and cell lines, we observed that expression of CXCR4 and DR5 are inversely correlated in human cancer. Together, we showed for the first time that targeting intracellular CXCR4 protein may be critical to dampen the pro-tumorigenic functions of CXCR4. Citation Format: Mushtaq Ahmad Nengroo, Shrankhla Maheshwari, Akhilesh Singh, Anup Kumar Singh, Priyank Chaturvedi, Ayushi Verma, Rakesh Kumar Arya, Krishan Kumar Saini, Annapurna Gupta, Anjali Mishra, Dipak Datta. CXCR4 intracellular protein regulates chemotherapy resistance by modulating the expression of death receptor 5 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6325.

In and Out of the Bursa-The Role of CXCR4 in Chicken B Cell Development.

In contrast to mammals, early B cell differentiation and diversification of the antibody repertoire in chickens do not take place in the bone marrow but in a specialized gut associated lymphoid tissue (GALT), the bursa of Fabricius. During embryonic development, B cell precursors migrate to the bursa anlage, where they proliferate and diversify their B cell receptor repertoire. Around hatch these diversified B cells start to emigrate from the bursa of Fabricius and populate peripheral lymphoid organs, but very little is known how the migratory processes are regulated. As CXCL12 (syn. SDF-1) and CXCR4 were shown to be essential for the control of B cell migration during the development of lymphoid tissues in mammals, we analyzed expression and function of this chemokine/chemokine-receptor pair in the chicken bursa. We found a strong variation of mRNA abundance of CXCL12 and CXCR4 in different stages of bursa development, with high abundance of CXCL12 mRNA in the bursa anlage at embryonic day 10 (ED10). In situ hybridization demonstrated disseminated CXCL12 expression in the early bursa anlage, which condensed in the developing follicles and was mainly restricted to the follicle cortex post-hatch. Flow cytometric analysis detected CXCR4 protein already on early B cell stages, increasing during bursal development. Post-hatch, a subpopulation with the hallmarks of emigrating B cells became detectable, which had lower CXCR4 expression, suggesting that downregulation of CXCR4 is necessary to leave the CXCL12-high bursal environment. In vivo blockade of CXCR4 using AMD3100 at the time of B cell precursor immigration strongly inhibited follicle development, demonstrating that CXCL12 attracts pre-bursal B cells into the bursal anlage. Altogether, we show that CXCL12 and its receptor CXCR4 are important for both populating the bursa with B cells and emigration of mature B cells into the periphery post hatch, and that CXCR4 function in primary B cell organs is conserved between mammals and birds.

Intravital Imaging Reveals Motility of Adult Hematopoietic Stem Cells in the Bone Marrow Niche

Adult mammalian hematopoietic stem cells (HSCs) reside in the bone marrow (BM) but can be mobilized into blood for use in transplantation. HSCs interact with BM niche cells that produce growth factor c-Kit ligand (Kitl/SCF) and chemokine CXCL12, and were thought to be static and sessile. We used two-photon laser scanning microscopy to visualize genetically labeled HSCs in the BM of live mice for several hours. The majority of HSCs showed a dynamic non-spherical morphology and significant motility, undergoing slow processive motion interrupted by short stretches of confined motion. HSCs moved in the perivascular space and showed intermittent close contacts with SCF-expressing perivascular stromal cells. In contrast, mobilization-inducing blockade of CXCL12 receptor CXCR4 and integrins rapidly abrogated HSC motility and shape dynamics in real time. Our results reveal an unexpectedly dynamic nature of HSC residence in the BM and interaction with the SCF+ stromal niche, which is disrupted during HSC mobilization.

SUN-164 CXCR4-AngII axis plays an important role in glomerular injury through promoting the crosstalk between podocytes and mesangial cells

Glomerular disease has a high incidence worldwide. Podocyte injury and mesangial cell activation play pivotal roles in the progression of proteinuria and glomerulosclerosis. However, the linkage and underlying mechanisms are rarely reported. Herein, we investigated the role of chemokine receptor (CXCR4) and angiotensin II (AngII) in the pathogenesis of podocyte and mesangial cell injury. The association of CXCR4-AngII axis in crosstalk between podocytes and mesangial cells was also testified. 5/6 nephrectomized mice was established. To identify the role of CXCR4, CXCR4 plasmid was injected once a week by tail injection. Some mice were injected with adriamycin (ADR), advanced oxidation protein products (AOPPs) or Losartan. In vitro, MPC5 cells, an immortalized mouse podocyte cell line, were incubated with AOPPs or transfected with CXCR4 expression plasmid or siRNA to CXCR4. RMC cells, an immortalized mesangial cell line, were also adopted. RMC were treated with the conditional medium from AOPPs-treated MPC5 cells to assess the crosstalk between podocytes and mesangial cells. In 5/6NX and ADR mice model, CXCR4 was upregulated predominantly in podocytes. Whereas, SDF-1α, the ligand of CXCR4, was highly expressed in mesangial cells. CXCR4 colocalized with AngII in podocytes. Administration of AOPPs significantly induced the upregulation of CXCR4 and accelerated the progression of proteinuria. AOPPs induced podocyte injury and mesangial cell proliferation. These glomerular lesions were accompanied by upregulation of CXCR4, SDF-1α and activation of ACE, Renin and AT1. In ADR model, ectopic expression of CXCR4 aggravated proteinuria, podocyte injury, mesangial cell proliferation and glomerulosclerosis. Treatment with losartan significantly ameliorated these pathological changes. In cultured MPC5 cells, incubation of AOPPs or overexpression of CXCR4 promoted the activation of ACE, Renin, and AT1. However, blockade of CXCR4 reduced these effects. In RMC cells, administration of AngII stimulated fibrogenesis and cell proliferation. The conditional medium from AOPPs-treated MPC5 cells induced upregulation of fibronectin, PCNA and angiotensin II receptor AT1, but blocked by incubation with losartan. Our studies indicate that CXCR4 could upregulate the expression of RAS system in podocyte. AngII may subsequently bind to its receptor AT1 in mesangial cells to induce matrix deposition. The activation of mesangial cells would lead to the secretion of SDF-1α, the ligand of CXCR4, which further triggers podocyte injury. These studies demonstrate that CXCR4-AngII axis plays a vital role in glomerular injury via mediating pathologic crosstalk between podocytes and mesangial cells. Our findings uncover a new pathogenic mechanism by which CXCR4-AngII axis promotes glomerulosclerosis.

CXCR4 inhibition modulates the tumor microenvironment and retards the growth of B16-OVA melanoma and Renca tumors.

To determine whether blockade of the chemokine receptor CXCR4 might alter the tumor microenvironment and inhibit tumor growth, we tested the efficacy of the CXCR4 antagonist X4-136 as a single agent and in combination with various immune checkpoint inhibitors in the syngeneic murine melanoma model B16-OVA. We also tested its activity alone and in combination with axitinib in the renal cancer model Renca. We found that X4-136 exhibited potent single agent antitumor activity in the B16-OVA model that was additive to that of an anti-PDL1 antibody. The antitumor activities were associated with a reduction in the number of immunosuppressive regulatory T cells and myeloid-derived suppressor cells and an increase in the number of tumor-specific CD8/perforin cells in the tumor-microenvironment. Apart from these immune effects, X4-136 alone and in combination with checkpoint inhibitors inhibited the Akt/FOXO-3a cell survival pathway in vitro and in vivo, suggesting that it might have antitumor activity independent of its effects on immune cell trafficking. Similar effects on tumor growth and cytotoxic T lymphocytes infiltration were observed in the Renca model. These studies show that the effects of CXCR4 blockade on immune cell trafficking might serve as a useful adjunct to immune checkpoint inhibitors and other therapies in the treatment of cancer.

Preexisting malignancy abrogates the beneficial effects of CXCR4 blockade during sepsis.

Patients with cancer are at an increased risk of developing and dying from sepsis. We previously reported that blockade of the chemokine receptor CXCR4 resulted in decreased CD4+ T cell exhaustion and improved survival in a model of polymicrobial sepsis in previously healthy mice. Here, we sought to determine whether CXCR4 blockade could improve mortality and immune dysregulation during sepsis complicated with malignancy. Results in animals inoculated with a lung cancer cell line and subjected to CLP 3 weeks later indicated that CXCR4 was up-regulated on naïve and central memory T cells following sepsis. Of note, and in contrast to results in previously healthy mice, CXCR4 blockade failed to improve survival in cancer septic animals; instead, it actually significantly worsened survival. In the setting of cancer, CXCR4 blockade failed to result in T cell egress from the bone marrow, reverse lymphopenia in the spleen, or reverse T cell exhaustion. Mechanistically, elevated expression of CD69 on naïve T cells in the bone marrow of cancer septic animals was associated with their inability to egress from the bone marrow in the setting of CXCR4 blockade. In conclusion, these results illuminate the differential impact of CXCR4 blockade on sepsis pathophysiology in the setting of cancer and highlight the need for personalized therapy during sepsis.

Pre-metastatic niche triggers SDF-1/CXCR4 axis and promotes organ colonisation by hepatocellular circulating tumour cells via downregulation of Prrx1

BackgroundCirculating tumour cells (CTCs), especially mesenchymal CTCs, are important determinants of metastasis, which leads to most recurrence and mortality in hepatocellular carcinoma (HCC). However, little is known about the underlying mechanisms of CTC colonisation in pre-metastatic niches.MethodsDetection and classification of CTCs in patients were performed using the CanPatrol™ system. A lentiviral vector expressing Prrx1-targeting shRNA was constructed to generate a stable HCC cell line with low expression of Prrx1. The effect of Prrx1 knockdown on stemness, migration, and drug resistance of the cell line was assessed, including involvement of SDF-1/CXCR4 signalling. Promising clinical applications of an inhibitor of STAT3 tyrosine phosphorylation, C188–9, and specific blockade with CXCR4 antibody were explored.ResultsThe number of mesenchymal CTCs in blood was closely associated with tumour recurrence or metastasis. Pre-metastatic niche-derived SDF-1 could downregulate Prrx1, which induced the stemness, drug resistance, and increased expression of CXCR4 in HCC cells through the STAT3 pathway in vitro. In vivo, mice bearing tumours of Prrx1 low-expressing cells had significantly shorter survival. In xenograft tumours and clinical samples, loss of Prrx1 was negatively correlated with increased expression of CXCR4 in lung metastatic sites compared with that in the primary foci.ConclusionsThese findings demonstrate that decreased expression of Prrx1 stimulates SDF-1/CXCR4 signalling and contributes to organ colonisation with blood CTCs in HCC. STAT3 inhibition and specific blockade of CXCR4 have clinical potential as therapeutics for eliminating organ metastasis in advanced HCC.

CXCR4 Blockade By BL-8040 in T Cell Acute Lymphoblastic Leukemia Decreases Mitochondrial Mass and Induces Non-Apoptotic Cell Death

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 10-15% of pediatric and 25% of adult ALL cases. CXCL12 is a CXC chemokine that is constitutively expressed at high levels in the bone marrow. CXCR4 is the major receptor for CXCL12 and is by far the most highly expressed chemokine receptor on T-ALL cells. Two groups recently showed that genetic loss of CXCR4 signaling in murine or human T-ALL cells markedly suppressed their growth in vivo. We previously reported that BL-8040, a potent new CXCR4 antagonist with sustained receptor occupancy, is active as monotherapy against T-ALL in mice. Indeed, a 2-week course of daily BL-8040 resulted in a median reduction in tumor burden of 32.1-fold (range 6.8 to 176) across 5 different T-ALL xenografts. Preliminary data from a clinical trial of BL-8040 plus nelarabine for relapsed T-ALL also suggest therapeutic activity, with a complete remission rate observed in 4/8 patients (50%), which compares favorably to published response rates of approximately 30% with single agent nelarabine. Here, we explore molecular mechanisms by which CXCR4 blockade induces T-ALL death. NOD-scid IL2Rgammanull (NSG) mice were injected with P12-Ichikawa cells, a T-ALL cell line modified to express click beetle red luciferase and GFP. Following T-ALL engraftment, mice were treated with a single dose of BL-8040, and then leukemic cells in the bone marrow harvested 24-48 hours later. Treatment with BL-8040 resulted in a marked suppression of Akt and Erk1/2 phosphorylation, suggesting that signaling through CXCR4 is the major source of PI3 kinase pathway activation in T-ALL cells. Surprisingly, treatment with BL-8040 did not affect cellular proliferation, as measured by Ki67/FxCycle Violet staining or by EdU labeling. Moreover, no increase in apoptosis, as measured by annexin V or activated caspase 3 expression, was observed. These data suggest that CXCR4 blockade induces a non-apoptotic cell death. To explore this possibility further, we performed transcriptome sequencing on T-ALL cells recovered from mice 24 hours after 1 dose of BL-8040. A total of 151 differentially expressed genes (FDR of < 0.05% and ≥ 2-fold change) were identified. Gene set enrichment analysis was strongly positive for alterations in oxidative phosphorylation, ribosome biogenesis, and carbohydrate metabolism. Ribosome function was assessed using O-propargyl-puromycin (OPP), which monitors global protein translation. No difference in global protein synthesis in T-ALL cells was observed after CXCR4 blockade in vivo. T-ALL cells are dependent on glutamine as a source of carbon, and PI3 kinase signaling positively regulates glutaminolysis. Thus, we hypothesized that CXCR4 blockade may induce T-ALL cell death by reducing glutamine metabolism. However, treatment of T-ALL cells in vitro with BL-8040 did not alter the cellular levels of glutamine or glutamate, as measured using a commercial bioluminescent assay. Confirmatory metabolic tracing studies using 13C-labeled glutamine and glucose are in progress. Finally, to explore the reduction in oxidative phosphorylation, we examined mitochondria function using Mitotracker Green. Treatment of T-ALL cells in vitro with BL-8040 for 24-48 hours induced a significant decrease in mitochondria number, suggesting induction of mitophagy. Collectively, these data suggest that T-ALL cells are addicted to CXCR4 signaling in vivo. CXCR4 blockade with BL-8040 induces a non-apoptotic cell death that is characterized by a loss of mitochondria. Disclosures Uy: Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Bohana-Kashtan:BiolineRx: Employment, Equity Ownership. Sorani:BiolineRx: Employment, Equity Ownership. Vainstein:BiolineRx: Employment, Equity Ownership.

Synergistic Targeting of BTK and E-Selectin/CXCR4 in the Microenvironment of Mantle Cell Lymphomas

Synergistic Targeting of BTK and E-Selectin/CXCR4 in the Microenvironment of Mantle Cell Lymphomas

CXCR4 Inhibition with BL-8040 in Combination with Nelarabine in Patients with Relapsed or Refractory T-Cell Acute Lymphoblastic Leukemia / Lymphoblastic Lymphoma

Background: The chemokine receptor, CXCR4, mediates the trafficking and retention of hematopoietic stem cells to the marrow through its interaction with the chemokine, CXCL12. CXCR4 is the most highly expressed chemokine receptor on T-ALL cells, and preclinical data demonstrate that CXCR4 is critical for the growth and survival of T-cell acute lymphoblastic lymphoma (T-ALL). Genetic loss or pharmacologic blockade of CXCR4 signaling suppresses T-ALL cell growth and leukemia initiating activity in murine models. BL-8040 is a novel synthetic peptide antagonist of the chemokine receptor, CXCR4. Compared to other CXCR4 inhibitors, BL-8040 is a more potent mobilizer of normal HSCs with sustained occupancy and inhibition of CXCR4. We previously reported that BL-8040 potently suppresses T-ALL cell growth in xenotransplantation models of T-ALL. To test the efficacy of BL-8040 in T-ALL by conducting a multicenter, open label pilot study of BL-8040 in combination with nelarabine in patients with relapsed or refractory disease (ClinicalTrials.gov: NCT0276338). Methods: Adults with relapsed or refractory T-ALL/LBL, age ≥18 years and ECOG PS ≤2, were eligible for the study. A peripheral blood lymphoblast count ≤50,000/mm3 was required prior to starting treatment. During cycle 1, subjects received daily subcutaneous administration of BL-8040 1.5 mg/kg from Day 1 to Day 6 with nelarabine 1,500 mg/m2 intravenously over 2 hours on Days 2, 4 and 6. In subsequent 21-day cycles, BL-8040 1.5 mg/kg was administered daily on days 1-5 with nelarabine 1,500 mg/m2 given on days 1, 3 and 5. Up to 4 cycles of treatment was allowed. Results: Nine patients with median age of 29 years (range 20-75) have been enrolled in the study out of which including fout patients were in untreated 1st relapse, one in 2nd relapse, two with relapsed and refractory disease, and two primary refractory patient. Two patients had failed prior allogeneic hematopoietic cell transplant. The rate of complete remission is 56% (5/9) with all responses occurring after 1 cycle of treatment. Adverse events occurred in 2 subjects requiring dose modification. Consistent with the known mechanism of BL-8040, one subject developed G3 leukocytosis after BL-8040 administration with the peripheral WBC increasing from 5.3 to 273 h/mm3 with evidence of spontaneous tumor lysis requiring interruption of BL-8040 and leukapheresis. A second patient developed injection site pain requiring omission of the final dose of BL-8040. Peripheral blood analyzed at baseline and 24 hours after the first dose of BL-8040 (prior to nelarabine) showed the following: 1) sustained blockade of CXCR4 on leukemic blasts; 2) mobilization of leukemic blasts into the blood; and 3) no induction of leukemia blast apoptosis as measured by activated caspase 3 expression. Conclusions: BL-8040 can be combined with nelarabine in subjects with relapsed or refractory T-ALL with encouraging CR rates. Evidence of on target effects on CXCR4 has been observed in this study with sustained CXCR4 receptor occupancy at 24 hours after administration along with tumor mobilization of ALL blasts into the peripheral circulation. Disclosures Uy: Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Kadia:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bioline RX: Research Funding; BMS: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding. Stock:Astellas: Membership on an entity's Board of Directors or advisory committees; Daiichi: Membership on an entity's Board of Directors or advisory committees; Research to Practice: Honoraria; UpToDate: Honoraria; Agios: Membership on an entity's Board of Directors or advisory committees; Kite, a Gilead Company: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Brammer:Verastem, Inc: Research Funding; Viracta Therapeutics, Inc.: Research Funding; Bioniz Therapeutics, Inc.: Research Funding. Bohana-Kashtan:BiolineRx: Employment, Equity Ownership. Vainstein:BiolineRx: Employment, Equity Ownership. Sorani:BiolineRx: Employment, Equity Ownership. Chen:BiolineRx: Employment. DiPersio:Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; Celgene: Consultancy; Amphivena Therapeutics: Consultancy, Research Funding; Magenta Therapeutics: Equity Ownership; NeoImmune Tech: Research Funding; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Incyte: Consultancy, Research Funding; Karyopharm Therapeutics: Consultancy. OffLabel Disclosure: BL-8040 for ALL

MA13.09 Cisplatin Sustains Lung Cancer Metastasis Through the Systemic Activation of SDF-1/CXCR4 Axis

Standard chemotherapy regimens have limited long-term efficacy in lung cancer patients due to chemoresistance and inefficacy in controlling metastatic disease. In pre-clinical models we have shown that cisplatin treatment enriches for the chemoresistant fraction of CD133+CXCR4+ lung cancer metastasis initiating cells (MICs), increasing distant metastasis development that can be prevented by CXCR4 blockade. Therefore, we hypothesize that the SDF-1/CXCR4 axis, implied in MICs maintenance/migration and in immune and stromal cells trafficking, could play a critical role in cisplatin-induced pro-metastatic effects. To study the effects of cisplatin in promoting a pre-metastatic niche, naïve SCID mice were treated with cisplatin plus/minus peptide R (5mg/kg), a novel inhibitor of CXCR4 and after 72h injected intravenously with metastatic H460 cell line. To assess the effect of the combination treatment in pre-clinical model, H460 subcutaneous xenografts were treated with cisplatin alone or with peptide R for three weeks. Content of MICs in xenografts, number and phenotype of lung metastasis and immune cells modulationt were evaluated by FACS and IHC We showed that cisplatin treatment of naïf SCID mice resulted in a rapid BM expansion of the subset of CCR2+CXCR4+Ly6Chigh inflammatory monocytes (IM), concomitantly with their recruitment to murine lungs guided by increased level of SFD-1 released by PDGFRβ+ stromal cells in response to cisplatin. Peptide R partially prevented these effects. Tail-vein injection of H460 human lung cancer cells 72h after cisplatin administration resulted in augmented number of lung metastases (p=0.003), that showed a 3.5-fold enrichment in CD133+CXCR4+ MICs (p=0.005) and increase of IM and derived macrophages. Pre-treatment with peptide R abolished these effects. We verified that the abundance of CXCR4+CCR2+IM together with increased endothelial permeability caused by cisplatin may favor tumor cells extravasations and expansion of MICs through SDF-1/CXCR4 axis activation which determined metastasis overgrowth. SDF-1 was also increased in cisplatin-treated subcutaneous H460 xenografts that expanded the subset of chemoresistant CD133+CXCR4+ MICs and recruited CXCR4+tumor associated macrophages which may allow MICs to escape primary tumor. At the metastatic site cisplatin treatment of H460 xenografts caused an increase in stromal SDF-1 and recruitment of both CXCR4+ inflammatory monocytes/macrophages (1.6-fold change p=0.01) and MICs subset (1.8-fold change p=0,04), overall resulting in a boost in micrometastases. CXCR4 inhibition prevented the co-recruitment and cross-talk of MICs and IM at distant site, counteracting the pro-metastatic effects of cisplatin. Matched case series of stage III chemo-naive NSCLC patients and cisplatin-based neo-adjuvant treated patients demonstrated a significant increased in SDF-1 after chemotherapy (p=0,0001). An high expression of tumoral SDF-1 (Score: staining intensity x % positive tumor cells >6) induced by cisplatin neo-adjuvant treatment was associated with a shorter DFS (p=0,0056) and poor OS (p=0,029). Our data reveal a paradoxical pro-metastatic effect of cisplatin that fosters MIC-IM recruitment and cross-talk via SDF-1/CXCR4 axis activation. A new combination strategy based on CXCR4 inhibition may disrupt these interactions, providing more effective and long-lasting results for lung cancer treatment

PTEN loss activates a functional AKT/CXCR4 signaling axis to potentiate tumor growth and lung metastasis in human osteosarcoma cells.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. Loss of the tumor suppressor PTEN or activation of chemokine receptor CXCR4 has been demonstrated to associate with OS respectively. However, the signaling mechanism underlying PTEN-mediated antitumor effect remains largely unknown, and the crosstalk between PTEN and CXCR4 in OS has not been investigated. Here, we uncover a PTEN/AKT/CXCR4 pathway nexus in highly tumorigenic and metastatic human 143B OS cells. Loss of PTEN activates AKT/CXCR4 signaling axis and regulates a series of tumor cell behaviors. Notably, ERK is inversely regulated by PTEN and its activation occurs downstream of AKT but upstream of CXCR4, suggesting this kinase to be an important mediator between AKT and CXCR4. In vivo studies show that overexpression of PTEN dramatically attenuates bone destruction, and this inhibition is associated with reduced CXCR4 expression in tumors. CXCR4 inhibitor AMD3100 also markedly suppresses tumor growth in the bone. In addition, PTEN overexpression or AMD3100 substantially inhibits tumor expansion in the lung. Our studies highlight a novel PTEN/AKT/CXCR4 signaling nexus in OS tumor growth and lung metastasis, and provide a strong rationale to consider PTEN restoration or CXCR4 blockade for the treatment of aggressive OS in humans.