Targeting the CXCR4–CXCL12 Axis—Untapped Potential in the Tumor

Author Manuscript Published OnlineFirst on July 21, 2015; DOI: 10.1158/1078-0432.CCR-14-0914 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Molecular Pathways: Targeting the CXCR4–CXCL12 Axis—Untapped Potential in the Tumor

Microenvironment

Stefania Scala

Molecular Immunology, Functional Genomics, Istituto Nazionale per lo Studio e la Cura dei

Tumori, “Fondazione G. Pascale,” IRCCS, Napoli, Italy.

Corresponding Author: Stefania Scala, Molecular Immunology, Functional Genomics, Istituto

Nazionale per lo Studio e la Cura dei Tumori, Fondazione “G. Pascale, ”IRCCS, Via Semmola,

80131, Naples, Italy. Phone: +39-081-5903678; Fax +39-081-5903820; E-mail: [email protected], [email protected]

Running Title: CXCR4 in Tumor Microenvironment

Disclosure of Potential Conflicts of Interest

S. Scala is listed as a co-inventor on a patent, which is co-owned by Istituto Nazionale per lo

Studio e la Cura dei Tumori and Consiglio Nazionale delle Ricerche, related to cyclic peptides binding CXCR4 receptor and relative medical and diagnostic uses. No other potential conflicts of interest were disclosed.

Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 21, 2015; DOI: 10.1158/1078-0432.CCR-14-0914 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Evidence suggests the CXC-Chemokine Receptor-4 pathway, plays a role in cancer cell homing and metastasis, and thus represents a potential target for cancer therapy. The homeostatic microenvironment chemokine, CXCL12, binds the CXCR4 and CXCR7 receptors, activating divergent signals on multiple pathways such as ERK1/2, p38, SAPK/JNK, AKT, mTOR and the

Bruton Tyrosine kinase (BTK). An activating mutation in CXCR4 is responsible for a rare disease,

WHIM syndrome, and dominant CXCR4 mutations have also been reported in Waldenstrom

Macroglobulinemia (WM). The CXCR4/CXCL12 axis regulates the hematopoietic stem cell niche - a property that has led to the approval of the CXCR4 antagonist, plerixafor (AMD3100), for mobilization of hematopoietic precursors. In preclinical models plerixafor has shown anti- metastatic potential in vivo, offering proof of concept. Other antagonists are in preclinical and clinical development. Recent evidence demonstrates that inhibiting CXCR4 signaling restores sensitivity to CTLA4 and PD-1 checkpoints inhibitors, creating new line for investigation.

Targeting the CXCR4-CXCL12 axis thus offers the possibility of affecting CXCR4-expressing primary tumor cells, modulating the immune response or synergizing with other targeted anticancer therapies.

Background

Chemokines are small chemo-attractant cytokines that are expressed in discrete anatomical locations. In adult vertebrates, chemokines are essential for proper lymphoid organ architecture and for leukocyte trafficking (1). Chemokines are classified according to their conserved N- terminal cysteine residues (C) that form the first disulfide bond. These residues can be adjacent

(CC) or separated by amino acid(s) (CXC, CX3C). Forty-seven chemokines have been identified -

27 CC and 17 CXC chemokines as well as a single CX3C and 2 single C chemokines (1,2).

Chemokines act on chemokine receptors (CKR), members of the seven transmembrane domain G protein coupled receptor (GPCR) superfamily. Classically, one of the chemokine receptor intracellular loops interacts with heterotrimeric, pertussis toxin-sensitive G proteins called Gαi, initiating a cascade of signal transduction events in response to ligand binding. In addition, chemokine receptors can signal through non-G protein-mediated pathways or even through other G-protein subtypes (3). There are also atypical chemokine receptors, (ACKR), which do not mediate conventional signaling and do not elicit directional migration (4,5); the CXCL12 – CXCR4 axis will be the focus of this Molecular Pathways review.

Encoded on chromosome 2q21, CXCR4 is an evolutionarily highly conserved GPCR expressed on monocytes, B cells and naive T cells in the peripheral blood. Human CXCR4 was originally identified as a receptor for CXCL12 by screening chemokine receptor orphan genes for their ability to induce intracellular Ca+2 in response to human CXCL12. Its ligand, CXCL12 is a homeostatic chemokine, which controls hematopoietic cell trafficking, adhesion, immune surveillance and development. The amino-terminal domain of CXCL12 binds the second extracellular loop of CXCR4 and activates downstream signaling pathways. The third intracellular loop of CXCR4 is necessary for Gαi-dependent signaling, and intracellular loops 2 and 3 as well as the C terminus of CXCR4 are required for chemotaxis (6,7).

CXCL12 binding to CXCR4 triggers multiple signal transduction pathways able to regulate intracellular calcium flux, chemotaxis, transcription and cell survival (8). CXCL12 binding promotes a three-dimensional CXCR4 conformation favoring Gαi protein dissociation into α- and βγ- subunits, (8,9). In turn, different subtypes of the α subunit impart different signals Gαi subunits inhibit cAMP formation via inhibition of adenylyl cyclase activity and the αq-subunits activate phospholipase C (PLC)-β, generating diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (IP3), which controls the release of intracellular Ca2+. While inhibiting adenilyl cyclase, the Gai subunits activate the NF-kB, JAK/STAT, and PI3K-AKT pathways as well as mTOR, and the JNK/p38 MAPKs regulating cell survival, proliferation, and chemotaxis. Recent studies have shown CXCR4 signaling through mTOR in pancreatic cancer, gastric cancer and T-cell leukemia cells (10-13). In human renal cancer cells, CXCL12 induces phosphorylation of the specific mTOR targets, P70S6K and 4EBP1 (14) and CXCR4 and mTOR inhibitors have been reported to impair human renal cancer migration (14) (Fig. 1). Unlike the α subunits, βγ dimer subunits promote RAS-mediated

MAPK signaling thereby regulating cell proliferation and chemotaxis (6). Finally, in addition to these classical signaling pathways, CXCR4 triggers Bruton Tyrosine Kinase (BTK) phosphorylation and downstream MAPK in mantle cell lymphoma and primary acute myeloid leukemia (AML) blasts, suggesting a potential interaction of CXCR4 on BTK and a potential for concomitant CXCR4 and BTK inhibition, the latter possibility raised by the availability of a recently approved inhibitor of BTK, ibrutinib (15).

CXCL12 binding to CXCR4 also causes CXCR4 desensitization, manifested as uncoupling from G-proteins by GPCR kinase (GRK)-dependent phosphorylation and subsequent interaction of

CXCR4 with β-arrestin, which mediates internalization of the receptor (16) (Fig. 1). Upon internalization, CXCR4 is targeted for lysosomal degradation (9, 16). This requires agonist-induced ubiquitination of the carboxyl terminal tail lysine residues (16).

Recently, CXCR7 was also found to be a high affinity receptor for CXCL12 and for CXCL11

(4, 17, 18). CXCR7, renamed as ACKR3, belongs to the atypical chemokine receptor group (ACKR) of proteins, which do not mediate conventional signaling and do not elicit directional migration.

The highly conserved DRYLAIV domain, which controls G-protein binding and activation in CXCR4, is replaced by DRYLSIT in the CXCR7 protein. Thus, typical chemokine responses mediated by G protein activity are not generated after ligand binding. However, recent evidence suggests that

CXCR7 activates intracellular signaling pathways, including the AKT, MAPK and JAK/STAT3 pathways (18). CXCR7 is also regulated by ubiquitination, but in contrast to CXCR4, CXCR7 is constitutively ubiquitinated and agonist-activation induces its de-ubiquitination. Upon agonist binding, CXCR7 is rapidly internalized via a β-arrestin-dependent pathway and recycled to the cell surface. CXCR7 itself is not degraded, but rather it delivers bound chemokine to lysosomes for degradation (16). β-arrestin recruitment to the CXCR4/CXCR7 complex enhances downstream β- arrestin-dependent cell signaling (ERK1/2, p38, SAPK/JNK), which induces cell migration in response to CXCL12 (11, 13, 18, 19).

Clinical–Translational Advances

The CXCR4 antagonist AMD3100 is the most studied among the agents that inhibit

CXCL12/CXCR4 signaling. AMD3100 was initially studied as an anti-HIV agent and then it was discovered that this compound increases white blood cell counts in the blood and is able to mobilize stem cells from the bone marrow (20, 21). Cell migration in and out of the bone marrow follows opposite chemokine signals, which implies that chemokines regulate the numbers of circulating granulocytes and monocytes. CXCL12 is the predominant signal that retains CXCR4+ hematopoietic stem cells (HSCs) inside the bone marrow (21). Plerixafor (previously AMD3100) is available clinically, having been developed as a mobilizing agent for hematopoietic precursors.

The FDA approved plerixafor in 2008 for “use in combination with granulocyte-colony stimulating

factor (G-CSF) to mobilize hematopoietic stem cells to the peripheral blood for collection and subsequent autologous transplantation in patients with non-Hodgkin lymphoma (NHL) and multiple myeloma (MM)” (20, 22).

A clinical indication for CXCR4 antagonists has emerged recently in the rare human WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis). WHIM syndrome is associated with germ line dominant mutations in the C terminus of CXCR4, which result in a truncation of the receptor (23). This blocks receptor internalization, determining persistent

CXCR4 activation and bone marrow myeloid cell retention, an outcome known as myelokathexis, the retention and apoptosis of mature neutrophils in the bone marrow (24). Two Phase I trials showed that plerixafor was able to safely and rapidly increase absolute lymphocyte, monocyte, and neutrophil counts in the peripheral blood of WHIM syndrome patients in a dose-dependent manner for 1 to 2 weeks (25). Another Phase I study demonstrated a durable increase in circulating leukocytes, fewer infections and improvement in warts in combination with topical imiquimod in three patients with WHIM syndrome who self-injected a low dose of plerixafor (4% to 8% of the FDA-approved dose) subcutaneously twice daily for 6 months (26). However, despite the fact that B and T cells were mobilized to blood, and that naive B cells underwent class switching in vitro, there was not an improvement in Ig levels or vaccine responses (26). Thus it appears that alternate dosing regimens or a more long-lasting CXCR4 antagonist will be required to achieve more durable activity that may in turn provide more stable levels of immune cells in the blood to enhance adaptive immune function.

CXCR4 activating mutations have also been recently found in Waldenstrom macroglobulinemia (WM). WM is an incurable B-cell neoplasm characterized by accumulation of malignant lymphoplasmacytic cells (LPC) in the bone marrow (BM), lymph nodes, and spleen, with excess production of serum immunoglobulin-M (IgM) producing hyperviscosity, tissue

infiltration and autoimmune-related pathology. Activating somatic mutations have been found by whole genome sequencing in WM, including a locus in CXCR4 (27). In addition, approximately

90% to 95% of WM patients harbor an activating mutation in MYD88 (MYD88L265P) that promotes both the interleukin-1 receptor–associated kinase (IRAK) and Bruton’s tyrosine kinase (BTK), which in turn activate nuclear factor-kB-p65–dependent nuclear translocation and malignant cell growth. The location of the CXCR4 somatic mutations in the C-terminal domain of WM patients is similar to the location of the mutations observed in the germ line of patients with WHIM syndrome. Somatic mutations in MYD88 and CXCR4 are thus felt to be important and to impact overall survival in WM (27), opening the way for possible combination therapy directed against

MYD88 and CXCR4 signaling in WM (27).

The CXCR4-CXCL12 axis as a potential target in cancer therapy

While there has been great enthusiasm for exploiting the CXCR4-CXCL12 axis as a target in cancer therapy, to date the promise has yet to be fulfilled. Initial interest in pursuing the CXCR4-CXCL12 axis as a target for cancer therapy was fueled by observations implicating CXCR4 in promoting metastasis (2, 28). CXCR4 is expressed in at least 20 different human cancers (8, 29-33); CXCR7 is also expressed in tumors and found to be involved in cell growth, survival, and metastasis (34,

35). Like CXCR4, it is expressed on tumor-associated vessels and on neovasculature (36). On the other hand, CXCL12 is secreted in the tumor microenvironment by stromal cells (21, 28). Based on data such as these, it has been thought that CXCR4 antagonism could prevent the development of metastases by targeting multiple steps in the process of dissemination.

Restricted by the tumor type, inhibiting CXCR4 should interfere with tumor cell growth, migration and chemotaxis, and homing toward secondary organs.

In addition to a potential role for agents targeting the CXCR4-CXCL12 axis in preventing metastasis, other indications have been suggested. Some have proposed that inhibition of the

CXCR4-CXCL12 axis could be used to mobilize leukemic cells from the marrow, rendering them more amenable to cytotoxic chemotherapy by removing them from the pro-survival stem cell niche (8, 21, 37-39).

Others have suggested that modulation of the CXCR4-CXCL12 axis could revert the tolerogenic polarization of the microenvironment rich of immunosuppressive cells such as Treg,

M2 and N2 neutrophils (40-42). Recent data show that blocking the interaction of T-cells expressing CXCR4 with cells in the microenvironment secreting CXCL12 may modulate immunotherapy with anti-CTLA4 or anti-PD-1 (41, 42). Although inhibition checkpoints have been shown to induce immune-mediated tumor shrinkage, major responses have been reported in only a subset of patients following PD-1 blockade (43). The resistance to immunotherapy reported in colon, ovary cancer and in the model of pancreatic ductal adenocarcinoma (PDA) is based on multiple mechanisms: spatial distribution of effector T cells relatively to tumor, recruitment of tumor specific T cells from the vessel, and T cells proliferation activity. In the PDA model, cancer associated fibroblasts (CAF) cells may regulate the T cell access to the tumor through release of CXCL12 which is bound by the PDA cancer cells. Plerixafor, inhibiting CXCR4, increases accumulation of T cells among cancer cells impairing tumor growth and increasing tumor sensitivity to anti-PD-L1. The related mechanism must involve either T cells or myelomonocytic cells, CXCR4 expressing cells. Of note, in this model neither cancer cells nor CAF-

FAP+ cells express CXCR4 (41, 44). CXCR4-dependent modulation of immunotherapy was demonstrated in hepatocellular carcinoma (HCC) models. Sorafenib treatment of orthotopic HCC induced hypoxia responsible of development of resistance. Sorafenib resistance was mediated by increased presence of immunosuppressive infiltrates (F4/80, tumor associated macrophages

(TAMs), myeloid derived suppressive cells and Treg) and increased PD-L1 expression in multiple hematopoietic cells, including immunosuppressive cells. Since the recruitment of

immunosuppressive cell population was partially mediated by CXCL12-induced hypoxia, the efficacy of CXCR4 inhibition was evaluated. A combined treatment (sorafenib/Plerixafor/anti-PD-

1) showed the most pronounced delay in tumor growth probably mediated by increased intratumoral penetration and activation of CD8 T lymphocytes in HCC (42). Moreover, in mouse model of intraperitoneal papillary epithelial ovarian cancer, CXCR4 antagonism increased tumor cell apoptosis and necrosis, reduced intraperitoneal dissemination, and selectively reduced intratumoral FoxP3+ Treg cells (40). Consistently with these data, Plerixafor and the antagonistic

CXCR4 peptide R (45, 46) inhibit Treg suppressive activity in primary renal cancer specimens in which higher numbers of activated Tregs (expressing CTLA-4/CXCR-4/PD-1/ICOS) were detected.

A possible mechanism may involve the Treg-FOXP3 promoter.

Treg-FOXP3 activity is regulated post-translationally by histone/protein acetyltransferases and histone/protein deacetylases (HDACs). Pan-histone/protein deacetylase inhibitors (pan-

HDACi) have been shown to increase FOXP3 acetylation and DNA binding, enhance Treg production and suppressive activity, and have beneficial effects in the prevention and treatment of autoimmune disease and transplant rejection (47). Since CXCR4 signaling transduces also on

FOXP3 and HDACis upregulated CXCR4 mRNA expression (48) it is possible that CXCR4 modulation affects the acetylation status of FOXP3 promoter.

CXCR4 antagonists in development

Plerixafor has many positive attributes, including demonstrated anti-metastatic potential in preclinical studies; however, there is room for the development of additional agents targeting the

CXCR4-CXCL12 axis. CXCR4 inhibition was originally conceived as a strategy to block infection of

CD4+ T cells by HIV since CXCR4 functions as a co-receptor for T-tropic (X4) HIV virus entry (20).

During development as anti HIV drug, plerixafor displayed a lack of oral bioavailability and cardiotoxicity. Additionally, its toxicity profile is such that it limits long-term administration as

might be required for metastasis prevention therapy (49). Although a 6 months administration was tolerated in WHIM patients, doses utilized were 4% to 8% of the FDA-approved dose (26).

Finally, plerixafor lacks CXCR4 specificity since it also binds the other CXCL12 receptor, CXCR7, as an allosteric agonist (50). The ability of CXCR4 antagonists to induce stem cell mobilization raised questions whether the same treatment might mobilize cells from solid tumors that express

CXCR4. In a recent Phase 1 Study, the efficacy of the peptidic CXCR4 antagonist LY2510924 was concomitantly evaluated on CD34+ mobilization versus count of circulating tumor cells (CTC).

Although there was significant mobilization of CD34+ cells upon treatment with LY2510924, no apparent effect on CTC count was observed (51).

Table 1 lists CXCR4 antagonists in clinical development. The majority of clinical trials have focused on the mobilizing activity of CXCR4 antagonists and were conducted in multiple myeloma, acute myeloid leukemia and chronic lymphatic leukemia (Table 1). Among peptidic inhibitors, 4F-benzoyl-TN14003 (BKT-140) is a 14-residue bio-stable synthetic peptide, which binds CXCR4 with a greater affinity compared with plerixafor. Studies in mice demonstrated the efficient and superior mobilization and transplantation of stem cells collected with G-CSF-

BKT140, compared with the single agent alone (52, 53). Additional studies demonstrated anticancer activity (54) and an ability of BKT140 to directly induce cell death of chronic myeloid cells (CML) and to revert resistance to imatinib mediated by increasing BCL6 (37). A Phase I trial conducted in multiple myeloma patients showed that BKT140 was well-tolerated, and produced a robust mobilization that resulted in the collection of functional CD34+ cells which rapidly engrafted (55). However, to date, anti-cancer activity has not been demonstrated.

Evaluation of antimetastatic activity in solid tumors is in Phase I - II clinical development and a limited number of results are available. LY2510924 (Eli Lilly and Company®) is a potent selective peptide CXCR4 antagonist; a phase I trial showed that the most common drug-related

adverse events were fatigue, injection-site reaction and nausea. However its potential as an anticancer agent has yet to be established. In the phase I trial, the best response achieved was stable disease in nine patients (20%) (51). In two Phase II studies, one in renal cell carcinoma

[NCT01391130] and one in small cell lung cancer (SCLC) [NCT01439568] that evaluated the efficacy of LY2510924 in combination with sunitinib and carboplatin/etoposide, respectively, anti- tumor efficacy was not achieved. In the SCLC trial there was no improvement in outcome for chemotherapy-naïve patients with extensive stage disease - small cell lung cancer (ED-SCLC) when 20 mg LY2510924 was added to carboplatin and etoposide (56). This outcome was observed despite preclinical data that was supportive of an anti-tumor strategy. The latter included 1) the fact that SCLC cells have been shown to express high levels of CXCR4 and activation of CXCR4 could induce cell migration and adhesion to stromal cells that secrete

CXCL12, and this in turn could provide growth and drug resistance signals to the tumor cells; and

2) a previous study that reported CXCR4 antagonists disrupt the CXCR4-mediated adhesion of

SCLC cells to stromal cells, in turn sensitizing SCLC cells to cytotoxic drugs such as etoposide, by antagonizing cell adhesion-mediated drug resistance (39). ED-SCLC may not be an ideal target for development as the disease often presents with extensive primary disease and with metastases at diagnosis and one would expect a CXCR4 antagonist to be mostly active in primary steps of metastatic dissemination.

CTCE-9908, a CXCL12 N-terminus derived peptide that is a dimerized sequence of CXCL12 amino acids 1-8 has also undergone a phase I-II clinical trial (57). CTCE-9908 was well tolerated with the most common side effect reported being mild to moderate injection site irritation in the

5.0 mg/kg/day dose group. However, the magnitude of anti-tumor efficacy was at best very modest, with six patients (30%) with evaluable although not clinically meaningful stable disease after one cycle (one month) (58).

Finally, we have recently reported a new family of CXCR4 antagonists developed through a ligand-based approach (45, 46). A three-residue segment identified in CXCL12 that was similar to, but in reverse order, to a peculiar inhibitory chemokine secreted by herpes virus 8 (HHV8) vMIP-II was the starting point (59, 60). The motif Ar1-Ar2-R, where Ar is an aromatic residue, constitutes the core of a cyclic peptide library evaluated for anti-CXCR4 activity. Three peptides (R, S and I) identified as potent CXCR4 antagonists were demonstrated to reduce the development of metastases in in vivo models (45, 46). Recent studies also demonstrated that peptides R, S and I efficiently mobilize LY6G+ cells better and longer than AMD3100 and increase bone marrow cellularity, mainly of immature precursors indicating an active hematopoietic stimulation.

The question is how best to design trials that will circumvent the problem that CXCR4 antagonists may not reduce primary tumor growth in cancers such as ovarian and colorectal cancer, where both local and distant metastasis prevention would be important and where CXCR4 likely plays a role. One proposed model of clinical development would be to add it to chemotherapy, such as fluorouracil and oxaliplatin, which are used in neoadjuvant treatment of locally advanced rectal cancer. Such a setting identifies a time limited treatment and a rapid evaluation of results. Moreover another ideal setting could be represented by treatment of glioblastoma following primary surgery in which it is possible hypothesize that CXCR4 inhibition coupled to radiotherapy may have an additional effect on vasculogenesis ameliorating tumor control, as shown in preclinical studies (61).

Conclusion

Extensive pre-clinical data indicates that targeting the CXCR4-CXCL12 axis may have several beneficial actions including: 1) affecting CXCR4-expressing primary tumor cells; 2) synergizing with other cancer-targeted therapies; and 3) modulating the immune response. While any or all of these could be valuable anti-cancer strategies, clinical results to date have been disappointing.

Additional studies and studies with new agents will be required to determine whether inhibition of the CXCR4-CXCL12 axis may produce enough anticancer activity to be effective therapy in the complex setting of human cancer.

Acknowledgments

The author is deeply indebted to the architect Dr. Barbara Tartarone for help in constructing Fig.

Grant Support

S. Scala was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) IG 13192.

References

1. Anders HJ, Romagnani P, Mantovani A. Pathomechanisms: homeostatic chemokines in

health, tissue regeneration, and progressive diseases. Trends Mol Med 2014;20:154-65.

2. Zlotnik A, Burkhardt AM, Homey B. Homeostatic chemokine receptors and organ-specific

metastasis. Nat Rev Immunol 2011;11:597-606.

3. Rajagopal S, Rajagopal K, Lefkowitz RJ. Teaching old receptors new tricks: biasing seven-

transmembrane receptors. Nat Rev Drug Discov 2010;9:373-86.

4. Bachelerie F, Graham GJ, Locati M, Mantovani A, Murphy PM, Nibbs R, et al. New

nomenclature for atypical chemokine receptors. Nat Immunol 2014;15:207-8.

5. Rosenbaum DM, Rasmussen SG, Kobilka BK. The structure and function of G-protein-

coupled receptors. Nature 2009;459:356-63.

6. Roland J, Murphy BJ, Ahr B, Robert-Hebmann V, Delauzun V, Nye KE, et al. Role of the

intracellular domains of CXCR4 in SDF-1-mediated signaling. Blood 2003;101:399-406.

7. Crump MP, Gong JH, Loetscher P, Rajarathnam K, Amara A, Arenzana-Seisdedos F, et al.

Solution structure and basis for functional activity of stromal cell-derived factor-1;

dissociation of CXCR4 activation from binding and inhibition of HIV-1. EMBO J

1997;16:6996-7007.

8. Teicher BA, Fricker SP. CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin Cancer Res

2010;16:2927-31.

9. Cheng ZJ, Zhao J, Sun Y, Hu W, Wu YL, Cen B, et al. beta-arrestin differentially regulates

the chemokine receptor CXCR4-mediated signaling and receptor internalization, and this

implicates multiple interaction sites between beta-arrestin and CXCR4. J Biol Chem

2000;275:2479-85.

10. Munk R, Ghosh P, Ghosh MC, Saito T, Xu M, Carter A, et al. Involvement of mTOR in

CXCL12 mediated T cell signaling and migration. PLoS One 2011;6:e24667.

11. Chen G, Chen SM, Wang X, Ding XF, Ding J, Meng LH. Inhibition of chemokine (CXC motif)

ligand 12/chemokine (CXC motif) receptor 4 axis (CXCL12/CXCR4)-mediated cell migration

by targeting mammalian target of rapamycin (mTOR) pathway in human gastric

carcinoma cells. J Biol Chem 2012;287:12132-41.

12. Hashimoto I, Koizumi K, Tatematsu M, Minami T, Cho S, Takeno N, et al. Blocking on the

CXCR4/mTOR signalling pathway induces the anti-metastatic properties and autophagic

cell death in peritoneal disseminated gastric cancer cells. Eur J Cancer 2008;44:1022-9.

13. Weekes CD, Song D, Arcaroli J, Wilson LA, Rubio-Viqueira B, Cusatis G, et al. Stromal cell-

derived factor 1alpha mediates resistance to mTOR-directed therapy in pancreatic cancer.

Neoplasia 2012;14:690-701.

14. Ierano C, Santagata S, Napolitano M, Guardia F, Grimaldi A, Antignani E, et al. CXCR4 and

CXCR7 transduce through mTOR in human renal cancer cells. Cell Death Dis

2014;5:e1310.

15. Chang BY, Francesco M, De Rooij MF, Magadala P, Steggerda SM, Huang MM, et al. Egress

of CD19(+)CD5(+) cells into peripheral blood following treatment with the Bruton tyrosine

kinase inhibitor ibrutinib in mantle cell lymphoma patients. Blood 2013;122:2412-24.

16. Marchese A. Endocytic trafficking of chemokine receptors. Curr Opin Cell Biol 2014;27:72-

17. Balabanian K, Lagane B, Infantino S, Chow KY, Harriague J, Moepps B, et al. The

chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T

lymphocytes. J Biol Chem 2005;280:35760-6.

18. Singh AK, Arya RK, Trivedi AK, Sanyal S, Baral R, Dormond O, et al. Chemokine receptor

trio: CXCR3, CXCR4 and CXCR7 crosstalk via CXCL11 and CXCL12. Cytokine Growth Factor

Rev 2013;24:41-9.

19. Scala S, Giuliano P, Ascierto PA, Ierano C, Franco R, Napolitano M, et al. Human

melanoma metastases express functional CXCR4. Clin Cancer Res 2006;12:2427-33.

20. De Clercq E. The AMD3100 story: the path to the discovery of a stem cell mobilizer

(Mozobil). Biochem Pharmacol 2009;77:1655-64.

21. Rettig MP, Ansstas G, DiPersio JF. Mobilization of hematopoietic stem and progenitor

cells using inhibitors of CXCR4 and VLA-4. Leukemia 2012;26:34-53.

22. Devine SM, Flomenberg N, Vesole DH, Liesveld J, Weisdorf D, Badel K, et al. Rapid

mobilization of CD34+ cells following administration of the CXCR4 antagonist AMD3100 to

patients with multiple myeloma and non-Hodgkin's lymphoma. J Clin Oncol

2004;22:1095-102.

23. Hernandez PA, Gorlin RJ, Lukens JN, Taniuchi S, Bohinjec J, Francois F, et al. Mutations in

the chemokine receptor gene CXCR4 are associated with WHIM syndrome, a combined

immunodeficiency disease. Nat Genet 2003;34:70-4.

24. Kallikourdis M, Trovato AE, Anselmi F, Sarukhan A, Roselli G, Tassone L, et al. The CXCR4

mutations in WHIM syndrome impair the stability of the T-cell immunologic synapse.

Blood 2013;122:666-73.

25. McDermott DH, Liu Q, Ulrick J, Kwatemaa N, Anaya-O'Brien S, Penzak SR, et al. The CXCR4

antagonist plerixafor corrects panleukopenia in patients with WHIM syndrome. Blood

2011;118:4957-62.

26. McDermott DH, Liu Q, Velez D, Lopez L, Anaya-O'Brien S, Ulrick J, et al. A phase 1 clinical

trial of long-term, low-dose treatment of WHIM syndrome with the CXCR4 antagonist

plerixafor. Blood 2014;123:2308-16.

27. Treon SP, Cao Y, Xu L, Yang G, Liu X, Hunter ZR. Somatic mutations in MYD88 and CXCR4

are determinants of clinical presentation and overall survival in Waldenstrom

macroglobulinemia. Blood 2014;123:2791-6.

28. Balkwill F. Cancer and the chemokine network. Nat Rev Cancer 2004;4:540-50.

29. D'Alterio C, Avallone A, Tatangelo F, Delrio P, Pecori B, Cella L, et al. A prognostic model

comprising pT stage, N status, and the chemokine receptors CXCR4 and CXCR7 powerfully

predicts outcome in neoadjuvant resistant rectal cancer patients. Int J Cancer

2014;135:379-90.

30. D'Alterio C, Cindolo L, Portella L, Polimeno M, Consales C, Riccio A, et al. Differential role

of CD133 and CXCR4 in renal cell carcinoma. Cell Cycle 2010;9:4492-500.

31. Polimeno M, Ierano C, D'Alterio C, Simona Losito N, Napolitano M, Portella L, et al. CXCR4

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

Cell Mol Immunol 2015;12:474-82.

32. Ottaiano A, Franco R, Aiello Talamanca A, Liguori G, Tatangelo F, Delrio P, et al.

Overexpression of both CXC chemokine receptor 4 and vascular endothelial growth factor

proteins predicts early distant relapse in stage II-III colorectal cancer patients. Clin Cancer

Res 2006;12:2795-803.

33. Scala S, Ottaiano A, Ascierto PA, Cavalli M, Simeone E, Giuliano P, et al. Expression of

CXCR4 predicts poor prognosis in patients with malignant melanoma. Clin Cancer Res

2005;11:1835-41.

34. Miao Z, Luker KE, Summers BC, Berahovich R, Bhojani MS, Rehemtulla A, et al. CXCR7

(RDC1) promotes breast and lung tumor growth in vivo and is expressed on tumor-

associated vasculature. Proc Natl Acad Sci U S A 2007;104:15735-40.

35. Hattermann K, Held-Feindt J, Lucius R, Muerkoster SS, Penfold ME, Schall TJ, et al. The

chemokine receptor CXCR7 is highly expressed in human glioma cells and mediates

antiapoptotic effects. Cancer Res 2010;70:3299-308.

36. Liu Y, Carson-Walter E, Walter KA. Chemokine receptor CXCR7 is a functional receptor for

CXCL12 in brain endothelial cells. PLoS One 2014;9:e103938.

37. Beider K, Darash-Yahana M, Blaier O, Koren-Michowitz M, Abraham M, Wald H, et al.

Combination of imatinib with CXCR4 antagonist BKT140 overcomes the protective effect

of stroma and targets CML in vitro and in vivo. Mol Cancer Ther 2014;13:1155-69.

38. Duda DG, Kozin SV, Kirkpatrick ND, Xu L, Fukumura D, Jain RK. CXCL12 (SDF1alpha)-

CXCR4/CXCR7 pathway inhibition: an emerging sensitizer for anticancer therapies? Clin

Cancer Res 2011;17:2074-80.

39. Burger JA, Peled A. CXCR4 antagonists: targeting the microenvironment in leukemia and

other cancers. Leukemia 2009;23:43-52.

40. Righi E, Kashiwagi S, Yuan J, Santosuosso M, Leblanc P, Ingraham R, et al. CXCL12/CXCR4

blockade induces multimodal antitumor effects that prolong survival in an

immunocompetent mouse model of ovarian cancer. Cancer Res 2011;71:5522-34.

41. Feig C, Jones JO, Kraman M, Wells RJ, Deonarine A, Chan DS, et al. Targeting CXCL12 from

FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1

immunotherapy in pancreatic cancer. Proc Natl Acad Sci U S A 2013;110:20212-7.

42. Chen Y, Ramjiawan RR, Reiberger T, Ng MR, Hato T, Huang Y, et al. CXCR4 inhibition in

tumor microenvironment facilitates anti-PD-1 immunotherapy in sorafenib-treated HCC

in mice. Hepatology 2015;61:1591-602.

43. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, et al. Safety,

activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med

2012;366:2443-54.

44. Joyce JA, Fearon DT. T cell exclusion, immune privilege, and the tumor microenvironment.

Science 2015;348:74-80.

45. Portella L, Vitale R, De Luca S, D'Alterio C, Ierano C, Napolitano M, et al. Preclinical

development of a novel class of CXCR4 antagonist impairing solid tumors growth and

metastases. PLoS One 2013;8:e74548.

46. Amodeo P, Vitale R, De Luca S, Scala S, Castello G, Siani A, inventors. Cyclic peptides

binding CXCR4 receptor and relative medical and diagnostic uses. Europe patent EP

2528936. 2014 Oct 22.

47. Wang L, Liu Y, Han R, Beier UH, Bhatti TR, Akimova T, et al. FOXP3+ regulatory T cell

development and function require histone/protein deacetylase 3. J Clin Invest

2015;125:1111-23.

48. Ierano C, Basseville A, To KK, Zhan Z, Robey RW, Wilkerson J, et al. Histone deacetylase

inhibitors induce CXCR4 mRNA but antagonize CXCR4 migration. Cancer Biol Ther

2013;14:175-83.

49. Hendrix CW, Collier AC, Lederman MM, Schols D, Pollard RB, Brown S, et al. Safety,

pharmacokinetics, and antiviral activity of AMD3100, a selective CXCR4 receptor inhibitor,

in HIV-1 infection. J Acquir Immune Defic Syndr 2004;37:1253-62.

50. Kalatskaya I, Berchiche YA, Gravel S, Limberg BJ, Rosenbaum JS, Heveker N. AMD3100 is a

CXCR7 ligand with allosteric agonist properties. Mol Pharmacol 2009;75:1240-7.

51. Galsky MD, Vogelzang NJ, Conkling P, Raddad E, Polzer J, Roberson S, et al. A phase I trial

of LY2510924, a CXCR4 peptide antagonist, in patients with advanced cancer. Clin Cancer

Res 2014;20:3581-8.

52. Abraham M, Biyder K, Begin M, Wald H, Weiss ID, Galun E, et al. Enhanced unique pattern

of hematopoietic cell mobilization induced by the CXCR4 antagonist 4F-benzoyl-TN14003.

Stem Cells 2007;25:2158-66.

53. Abraham M, Beider K, Wald H, Weiss ID, Zipori D, Galun E, et al. The CXCR4 antagonist 4F-

benzoyl-TN14003 stimulates the recovery of the bone marrow after transplantation.

Leukemia 2009;23:1378-88.

54. Tamamura H, Hori A, Kanzaki N, Hiramatsu K, Mizumoto M, Nakashima H, et al. T140

analogs as CXCR4 antagonists identified as anti-metastatic agents in the treatment of

breast cancer. FEBS Lett 2003;550:79-83.

55. Peled A, Abraham M, Avivi I, Rowe JM, Beider K, Wald H, et al. The high-affinity CXCR4

antagonist BKT140 is safe and induces a robust mobilization of human CD34+ cells in

patients with multiple myeloma. Clin Cancer Res 2014;20:469-79.

56. Spigel DR, Weaver RW, McCleod M, Hamid O, Stille JR, Polzer J, et al. Phase II study of

carboplatin/etoposide plus LY2510924, a CXCR4 peptide antagonist, versus

carboplatin/etoposide in patients with extensive-stage small cell lung cancer (SCLC). Ann

Oncol 2014;25(suppl_4):iv511–6.

57. Wong D, Kandagatla P, Korz W, Chinni SR. Targeting CXCR4 with CTCE-9908 inhibits

prostate tumor metastasis. BMC Urol 2014;14:12.

58. Hotte SJ, Hirte HW, Moretto P, Iacobucci A, Wong D, Korz W, et al. Final results of a Phase

I/II study of CTCE-9908, a novel anticancer agent that inhibits CXCR4, in patients with

advanced solid cancers. Eur J Cancer Suppl 2008;6:127.

59. Crump MP, Elisseeva E, Gong J, Clark-Lewis I, Sykes BD. Structure/function of human

herpesvirus-8 MIP-II (1-71) and the antagonist N-terminal segment (1-10). FEBS Lett

2001;489:171-5.

60. Kledal TN, Rosenkilde MM, Coulin F, Simmons G, Johnsen AH, Alouani S, et al. A broad-

spectrum chemokine antagonist encoded by Kaposi's sarcoma-associated herpesvirus.

Science 1997;277:1656-9.

61. Kioi M, Vogel H, Schultz G, Hoffman RM, Harsh GR, Brown JM. Inhibition of

vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after

irradiation in mice. J Clin Invest 2010;120:694-705.

62. Wong D, Korz W. Translating an Antagonist of Chemokine Receptor CXCR4: from bench to

bedside. Clin Cancer Res 2008;14:7975-80.

63. Faber A, Roderburg C, Wein F, Saffrich R, Seckinger A, Horsch K, et al. The many facets of

SDF-1alpha, CXCR4 agonists and antagonists on hematopoietic progenitor cells. J Biomed

Biotechnol 2007;2007:26065.

64. Ichiyama K, Yokoyama-Kumakura S, Tanaka Y, Tanaka R, Hirose K, Bannai K, et al. A

duodenally absorbable CXC chemokine receptor 4 antagonist, KRH-1636, exhibits a

potent and selective anti-HIV-1 activity. Proc Natl Acad Sci U S A 2003;100:4185-90.

Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2015 American Association for Cancer Research. Table 1. CXCR4 antagonists under clinical investigation Downloaded from Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited.

Name Company Description Amino Acid Sequences or Chemical Indication Author ManuscriptPublishedOnlineFirstonJuly21,2015;DOI:10.1158/1078-0432.CCR-14-0914 Structures Hematopoietic stem cell mobilization in NCT01339039 patients with non-Hodgkin's lymphoma and clincancerres.aacrjournals.org multiple myeloma. FDA approved Glioma Small organic Acute myeloid leukemia NCT01141543 AMD3100 Sanofi molecule/sc Chronic lymphocytic leukemia NCT01373229 Myelokathexis (WHIM syndrome) NCT00967785 Ewing's sarcoma NCT01288573 Neuroblastoma Brain Tumors

Research. AMD 11070 HIV infections /X4 tropic virus NCT00361101 Corp. molecule HIV infections NCT00063804

NCT005911682 Small organic Refractory metastatic or locally advanced MSX122 Emory Univ molecule tumors

Chemokine Peptide (62) CTCE-9908 Therapeutics CXCL12- KGVSLSYRKRYSLSVGK Solid tumors antagonist Corp. mimetic

Chemokine Peptide KPVSLSYRAPFRFF (63) CTCE-0214 Therapeutics CXCL12- -Linker- Mobilization BM recovery agonist Corp. mimetic LKWIQEYLEKALN

Peptide Renal cancer NCT01391130 LY2510924 Lilly CXCR4- SCLC antagonist NCT01439568

Downloaded from Multiple myeloma NCT01018979 TaiGen Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. Small Non-hodgkin's lymphoma NCT01458288 TG-0054 Biotechnolo Heterocyclic (structure not disclosed publicly) Author ManuscriptPublishedOnlineFirstonJuly21,2015;DOI:10.1158/1078-0432.CCR-14-0914 molecule Hodgkin's disease healthy NCT00822341 gy

Metastatic breast cancer NCT01837095 Hematologic malignancies NCT01413568 clincancerres.aacrjournals.org Small Multiple myeloma NCT01105403 POL6326 Polyphor Ltd Protein Epitope Mimetics (PEM) Technology molecule Large reperfused ST-elevation myocardial NCT01905475 infarction NCT01841476 Healthy volunteers Multiple myeloma NCT01010880 Biok.Ther. Chronic myeloid leukemia NCT02115672 BKT-140 Ltd Peptide 4F-benzoyl-TN14003 Acute myeloid leukemia NCT01838395 (BL-8040) BioLineRx (T140 analog) Mobilization of hematopoietic stem cells (HSC) NCT02073019 Ltd to peripheral blood (PB) Autologous stem cell transplantation NCT00976378 on October 2,2021. ©2015 American Association forCancer

Research. NOXXON Hematopoietic stem cell transplantation NCT01194934 NOX-A12 anti-CXCL12 L-enantiomeric RNA oligonucleotide (Spiegelmer) Pharma AG Multiple myeloma NCT01521533 Chronic lymphocytic leukemia NCT01486797 Acute myelogenous leukemia BMS- Bristol- Diffuse large B-cell leukemia anti-CXCR4 NCT01120457 936564 Myers IgG4 antibody Chronic lymphocytic leukemia antibody NCT01359657 (MDX-1338) Squibb Follicular lymphoma Multiple myeloma

ALX-0651 Ablynx anti-CXCR4 Nanobody Healthy volunteers NCT01374503

Kureha Small organic KRH-1636 Chemical Anti-HIV-1 activity (64) molecule Industry

Author Manuscript Published OnlineFirst on July 21, 2015; DOI: 10.1158/1078-0432.CCR-14-0914 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 1. CXCR4-CXCL12-CXCR7 transduction pathway. CXCL12 acts on two distinct receptors, CXCR4 and

CXCR7, which are 7 membrane transpanning receptors/G protein coupled (GPCRs). CXCR4 and CXCR7 can form homodimers or heterodimers. CXCL12 shares CXCR7 binding with another CK, CXCL11 that is also a ligand for CXCR3. CXCR4 triggers preferentially G-protein-coupled signaling, whereas activation of CXCR7 or the CXCR4/CXCR7 complex induces β-arrestin-mediated signaling. The Gαi monomer inhibits the adenylyl cyclase activity regulating cell survival, proliferation and chemotaxis. While Gαi triggers

PI3K/AKT/mTOR and ERK1/2, Gβγ dimer triggers intracellular calcium mobilization through phospholipase

C (PLC). When CXCL12 binds CXCR7, the receptor signals through β-arrestin, inhibits G-protein-coupled signaling and activates MAP kinase pathway. CXCR7 can also signal through PLC/MAPK to increase cell survival. CXCR4–CXCR7 heterodimers-β-arrestin pathway can be activated through GPCR kinase (GRK)- dependent phosphorylation to internalize CXCR4, scavenging CXCL12 or/and control cell survival through

ERK1/2. CXCL12 also causes CXCR4 desensitization, uncoupling from G-protein by GRK-dependent phosphorylation and β-arrestin-dependent endocytosis. In contrast to CXCR4, when CXCL12 binds CXCR7 the interaction between β-arrestin and CXCR7 internalizes the receptor, and subsequent recycles it to the cell membrane. Upon binding to CXCR4 or CXCR7, CXCL12 is internalized and subjected to lysosomal degradation. Activator signals _____; Inhibitory signals ......

Figure 1:

CXCL12 CXCL11

CXCR4 CXCR7 CXCR3

CXCR4/7

B B GRK B GRK GRK A A A G G G

B-arrestin PLCB-arrestin B-arrestin PLC PI3KRAS AC

PIP2 PDK1 RAF cAMP ERK1/2

IP3 AKT PKA ERK1/2

Ca2+ mTOR Lysosome Raptor

P70S6K 4EBP1

Cell CXCR4 Cell CXCL12 CXCL12 Migration, Proliferation, Proliferation, survival, endocytosis, survival, scavenging scavenging chemotaxis chemotaxis chemotaxis proliferation desensitization proliferation

Molecular Pathways: Targeting the CXCR4-CXCL12 Axis-Untapped Potential in the Tumor Microenvironment

Stefania Scala

Clin Cancer Res Published OnlineFirst July 21, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-14-0914

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2015/07/18/1078-0432.CCR-14-0914. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.