Targeting the CXCR4– CXCL12 Axis—Untapped Potential in the Tumor Microenvironment Stefania Scala

Published OnlineFirst July 21, 2015; DOI: 10.1158/1078-0432.CCR-14-0914

Molecular Pathways Clinical Cancer Research Molecular Pathways: Targeting the CXCR4– CXCL12 Axis—Untapped Potential in the Tumor Microenvironment Stefania Scala

Abstract

Evidence suggests that the CXC–chemokine receptor-4 path- property that has led to the approval of the CXCR4 antagonist way plays a role in cancer cell homing and metastasis, and thus plerixafor (AMD3100) for mobilization of hematopoietic pre- represents a potential target for cancer therapy. The homeo- cursors. In preclinical models, plerixafor has shown antimetastatic microenvironment chemokine CXCL12 binds the CXCR4 static potential in vivo, offering proof of concept. Other antago- and CXCR7 receptors, activating divergent signals on multiple nists are in preclinical and clinical development. Recent evi- pathways, such as ERK1/2, p38, SAPK/JNK, AKT, mTOR, and dence demonstrates that inhibiting CXCR4 signaling restores the Bruton tyrosine kinase (BTK). An activating mutation in sensitivity to CTLA-4 and PD-1 checkpoint inhibitors, creating CXCR4 is responsible for a rare disease, WHIM syndrome a new line for investigation. Targeting the CXCR4–CXCL12 (warts, hypogammaglobulinemia, infections, and myelo- axis thus offers the possibility of affecting CXCR4-expressing kathexis), and dominant CXCR4 mutations have also been primary tumor cells, modulating the immune response, or reported in Waldenstrom macroglobulinemia. The CXCR4– synergizing with other targeted anticancer therapies. Clin Cancer CXCL12 axis regulates the hematopoietic stem cell niche—a Res; 21(19); 1–8. 2015 AACR.

Background na€ve T cells in the peripheral blood. Human CXCR4 was originally identified as a receptor for CXCL12 by screening CKR Chemokines are small chemoattractant cytokines that are þ orphan genes for their ability to induce intracellular Ca 2 in expressed in discrete anatomical locations. In adult vertebrates, response to human CXCL12. Its ligand, CXCL12, is a homeostatic chemokines are essential for proper lymphoid organ architecture chemokine, which controls hematopoietic cell trafficking, adhe- and for leukocyte trafficking (1). Chemokines are classified sion, immune surveillance, and development. The amino-termi- according to their conserved N-terminal cysteine residues (C) nal domain of CXCL12 binds the second extracellular loop of that form the first disulfide bond. These residues can be adjacent CXCR4 and activates downstream signaling pathways. The third (CC) or separated by amino acids (CXC and CX3C). Forty-seven intracellular loop of CXCR4 is necessary for Gai-dependent chemokines have been identified—27 CC and 17 CXC chemo- signaling, and intracellular loops 2 and 3 as well as the C-terminus kines as well as a single CX3C and two single C chemokines (1, 2). of CXCR4 are required for chemotaxis (6, 7). Chemokines act on chemokine receptors (CKR), members of the CXCL12 binding to CXCR4 triggers multiple signal transduc- seven-transmembrane domain G-protein–coupled receptor tion pathways that are able to regulate intracellular calcium flux, (GPCR) superfamily. Classically, one of the CKR intracellular chemotaxis, transcription, and cell survival (8). CXCL12 binding loops interacts with heterotrimeric, pertussis toxin–sensitive G promotes a three-dimensional CXCR4 conformation favoring proteins called Gai, initiating a cascade of signal transduction Gai protein dissociation into a and bg subunits (8, 9). In turn, events in response to ligand binding. In addition, CKRs can signal different subtypes of the a subunit impart different signals: Gai through non–G-protein–mediated pathways or even through subunits inhibit cAMP formation via inhibition of adenylyl other G-protein subtypes (3). There are also atypical CKRs cyclase activity, and the aq subunits activate phospholipase C (ACKR), which do not mediate conventional signaling and do (PLC)-b, generating diacylglycerol and inositol 1,4,5 trisphos- not elicit directional migration (4, 5). The CXCL12–CXCR4 axis is þ phate (IP3), which controls the release of intracellular Ca2 . the focus of this Molecular Pathways review. While inhibiting adenilyl cyclase, the Gai subunits activate the Encoded on chromosome 2q21, CXCR4 is an evolutionarily NF-kB, JAK–STAT, and PI3K–AKT pathways as well as mTOR, and highly conserved GPCR expressed on monocytes, B cells, and the JNK/p38 MAPKs regulating cell survival, proliferation, and chemotaxis. Recent studies have shown CXCR4 signaling through Molecular Immunology, Functional Genomics, Istituto Nazionale per lo mTOR in pancreatic cancer, gastric cancer, and T-cell leukemia Studio e la Cura dei Tumori, "Fondazione G. Pascale," IRCCS, Napoli, cells (10–13). In human renal cancer cells, CXCL12 induces Italy. phosphorylation of the specific mTOR targets, P70S6K and 4EBP1 Corresponding Author: Stefania Scala, Istituto Nazionale per lo Studio e la Cura (14), and CXCR4 and mTOR inhibitors have been reported to dei Tumori, Fondazione "G. Pascale," Via Semmola, Naples 80131, Italy. Phone: impair human renal cancer migration (Fig. 1; ref. 14). Unlike the a 39-081-590-3678; Fax 39-081-590-3820; E-mail: [email protected]; subunits, bg dimer subunits promote RAS-mediated MAPK sig- [email protected] naling, thereby regulating cell proliferation and chemotaxis (6). doi: 10.1158/1078-0432.CCR-14-0914 Finally, in addition to these classic signaling pathways, CXCR4 2015 American Association for Cancer Research. triggers Bruton tyrosine kinase (BTK) phosphorylation and

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CXCL12 CXCL11

CXCR4 CXCR7 CXCR3

CXCR4/7

β β GRK β GRK GRK α α α γ γ γ

β-arrestin PLCβ-arrestin β-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

Figure 1. The CXCR4–CXCL12–CXCR7 transduction pathway. CXCL12 acts on two distinct receptors, CXCR4 and CXCR7, which are seven-membrane transpanning receptors/ G-protein coupled (GPCR). CXCR4 and CXCR7 can form homodimers or heterodimers. CXCL12 shares CXCR7 binding with another chemokine, 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 b-arrestin– mediated signaling. The Gai monomer inhibits the adenylyl cyclase activity regulating cell survival, proliferation, and chemotaxis. Although Gai triggers PI3K/AKT/mTOR and ERK1/2, the Gbg dimer triggers intracellular calcium mobilization through PLC. When CXCL12 binds CXCR7, the receptor signals through b-arrestin, inhibits G-protein–coupled signaling, and activates the MAPK pathway. CXCR7 can also signal through PLC/MAPK to increase cell survival. The CXCR4–CXCR7 heterodimers–b-arrestin pathway can be activated through GRK-dependent phosphorylation to internalize CXCR4, scavenging CXCL12, and/or control cell survival through ERK1/2. CXCL12 also causes CXCR4 desensitization, uncoupling from G-protein by GRK-dependent phosphorylation, and b-arrestin–dependent endocytosis. In contrast with CXCR4, when CXCL12 binds CXCR7 the interaction between b-arrestin and CXCR7 internalizes the receptor, and subsequently recycles it to the cell membrane. Upon binding to CXCR4 or CXCR7, CXCL12 is internalized and subjected to lysosomal degradation. Activator signals are represented by straight lines. Inhibitory symbols are represented by dashed lines.

downstream MAPK in mantle cell lymphoma and primary acute for lysosomal degradation (9, 16). This requires agonist-induced myeloid leukemia (AML) blasts, suggesting a potential interaction ubiquitination of the carboxyl terminal (C-terminal) tail lysine of CXCR4 on BTK and a potential for concomitant CXCR4 and residues (16). BTK inhibition, the latter possibility raised by the availability of a Recently, CXCR7 was also found to be a high-afﬁnity receptor recently approved inhibitor of BTK, ibrutinib (15). for CXCL12 and CXCL11 (4, 17, 18). CXCR7, renamed as ACKR3, CXCL12 binding to CXCR4 also causes CXCR4 desensitization, belongs to the atypical CKR group (ACKR) of proteins, which do manifested as uncoupling from G proteins by GPCR kinase not mediate conventional signaling and do not elicit directional (GRK)-dependent phosphorylation and subsequent interaction migration. The highly conserved DRYLAIV domain, which con- of CXCR4 with b-arrestin, which mediates internalization of the trols G-protein binding and activation in CXCR4, is replaced receptor (Fig. 1; ref. 16). Upon internalization, CXCR4 is targeted by DRYLSIT in the CXCR7 protein. Thus, typical chemokine

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responses mediated by G-protein activity are not generated CXCR4-activating mutations have also been recently found in after ligand binding. However, evidence suggests that CXCR7 Waldenstrom macroglobulinemia. Waldenstrom macroglobuli- activates intracellular signaling pathways, including the AKT, nemia is an incurable B-cell neoplasm characterized by accumu- MAPK, and JAK–STAT3 pathways (18). CXCR7 is also regulated lation of malignant lymphoplasmacytic cells in the bone marrow, by ubiquitination, but in contrast with CXCR4, CXCR7 is con- lymph nodes, and spleen, with excess production of serum IgM stitutively ubiquitinated, and agonist activation induces its producing hyperviscosity, tissue inﬁltration, and autoimmune- deubiquitination. Upon agonist binding, CXCR7 is rapidly inter- related pathology. Activating somatic mutations have been found nalized via a b-arrestin–dependent pathway and recycled to the by whole-genome sequencing in Waldenstrom macroglobuline- cell surface. CXCR7 itself is not degraded, but rather it delivers mia, including a locus in CXCR4 (27). In addition, approximately bound chemokine to lysosomes for degradation (16). b-arrestin 90% to 95% of Waldenstrom macroglobulinemia patients harbor recruitment to the CXCR4–CXCR7 complex enhances down- an activating mutation in MYD88 (MYD88L265P) that promotes stream b-arrestin–dependent cell signaling (ERK1/2, p38, and both the interleukin-1 receptor–associated kinase (IRAK) and SAPK/JNK), which induces cell migration in response to CXCL12 BTK, which in turn activate NF-kB–p65-dependent nuclear trans- (11, 13, 18, 19). location and malignant cell growth. The location of the CXCR4 somatic mutations in the C-terminal domain of Waldenstrom Clinical–Translational Advances macroglobulinemia patients is similar to the location of the mutations observed in the germline of patients with WHIM The CXCR4 antagonist AMD3100 is the most studied among syndrome. Somatic mutations in MYD88 and CXCR4 are thus the agents that inhibit CXCL12/CXCR4 signaling. AMD3100 felt to be important and to affect overall survival in Waldenstrom was initially studied as an anti-HIV agent, and then it was macroglobulinemia (27), opening the way for possible combi- discovered that this compound increases white blood cell nation therapy directed against MYD88 and CXCR4 signaling in counts in the blood and is able to mobilize stem cells from Waldenstrom macroglobulinemia (27). the bone marrow (20, 21). Cell migration in and out of the bone marrow follows opposite chemokine signals, which The CXCR4–CXCL12 axis as a potential target in implies that chemokines regulate the numbers of circulating cancer therapy granulocytes and monocytes. CXCL12 is the predominant signal þ Although there has been great enthusiasm for exploiting that retains CXCR4 hematopoietic stem cells (HSC) inside the the CXCR4–CXCL12 axis as a target in cancer therapy, to date bone marrow (21). Plerixafor (previously AMD3100) is avail- the promise has yet to be fulﬁlled. Initial interest in pursuing the able clinically, having been developed as a mobilizing agent for CXCR4–CXCL12 axis as a target for cancer therapy was fueled by hematopoietic precursors. The FDA approved plerixafor in 2008 observations implicating CXCR4 in promoting metastasis (2, 28). for "use in combination with granulocyte-colony stimulating CXCR4 is expressed in at least 20 different human cancers (8, 29– factor (G-CSF) to mobilize hematopoietic stem cells to the 33); CXCR7 is also expressed in tumors and found to be involved peripheral blood for collection and subsequent autologous in cell growth, survival, and metastasis (34, 35). Like CXCR4, it is transplantation in patients with non-Hodgkin lymphoma expressed on tumor-associated vessels and on neovasculature (NHL) and multiple myeloma" (20, 22). (36). On the other hand, CXCL12 is secreted in the tumor A clinical indication for CXCR4 antagonists has emerged microenvironment by stromal cells (21, 28). On the basis of the recently in the rare human WHIM syndrome (warts, hypogam- data such as these, it has been thought that CXCR4 antagonism maglobulinemia, infections, and myelokathexis). WHIM syn- could prevent the development of metastases by targeting mul- drome is associated with germline-dominant mutations in the tiple steps in the process of dissemination. Restricted by the tumor C-terminus of CXCR4, which result in a truncation of the type, inhibiting CXCR4 should interfere with tumor cell growth, receptor (23). This blocks receptor internalization, determining migration and chemotaxis, and homing toward secondary organs. persistent CXCR4 activation and bone marrow myeloid cell In addition to a potential role for agents targeting the CXCR4– retention, an outcome known as myelokathexis, the retention CXCL12 axis in preventing metastasis, other indications have and apoptosis of mature neutrophils in the bone marrow (24). been suggested. Some researchers have proposed that inhibition Two phase I trials showed that plerixafor was able to safely and of the CXCR4–CXCL12 axis could be used to mobilize leukemic rapidly increase absolute lymphocyte, monocyte, and neutro- cells from the marrow, rendering them more amenable to cyto- phil counts in the peripheral blood of WHIM syndrome patients toxic chemotherapy by removing them from the prosurvival stem in a dose-dependent manner for 1 to 2 weeks (25). Another cell niche (8, 21, 37–39). phase I study demonstrated a durable increase in circulating Others have suggested that modulation of the CXCR4– leukocytes, fewer infections, and improvement in warts in CXCL12 axis could revert the tolerogenic polarization of the combination with topical imiquimod in 3 patients with WHIM microenvironment rich of immunosuppressive cells such as syndrome who self-injected a low dose of plerixafor (4% to 8% regulatory T cells (Treg), M2, and N2 neutrophils (40–42). of the FDA-approved dose) s.c. twice daily for 6 months (26). Recent data show that blocking the interaction of T cells However, despite the fact that B cells and T cells were mobilized expressing CXCR4 with cells in the microenvironment secret- to blood, and that na€ve B cells underwent class switching in vitro, ing CXCL12 may modulate immunotherapy with anti–CTLA- no improvement was seen in Ig levels or vaccine responses (26). 4oranti–PD-1 (41, 42). Although inhibition checkpoints Thus, it appears that alternate dosing regimens or a more long- have been shown to induce immune-mediated tumor shrink- lasting CXCR4 antagonist will be required to achieve more age, major responses have been reported in only a subset of durable activity that may in turn provide more stable levels of patients following PD-1 blockade (43). The resistance to immune cells in the blood to enhance adaptive immune immunotherapy reported in colon cancer, ovarian cancer, and function. in the model of pancreatic ductal adenocarcinoma (PDA)

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Table 1. CXCR4 antagonists under clinical investigation Company/ Amino acid sequences or Trial number/ Name university Description chemical structures Indication reference AMD3100 Sanoﬁ Small organic molecule HSC mobilization in NCT01339039 patients with NHL and multiple myeloma; FDA approved Glioma AML NCT01141543 Chronic lymphocytic NCT01373229 leukemia Myelokathexis (WHIM NCT00967785 syndrome) Ewing sarcoma NCT01288573 Neuroblastoma Brain tumors AMD 11070 Genzyme Corp. Small organic molecule HIV infections NCT00089466 HIV infections/X4 tropic NCT00361101 virus HIV infections NCT00063804

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

CTCE-9908 Chemokine Peptide CXCL12 mimetic KGVSLSYRKRYSLSVGK Solid tumors (62) antagonist Therapeutics Corp. CTCE-0214 Chemokine Peptide CXCL12 mimetic KPVSLSYRAPFRFF-Linker- Mobilization bone marrow (63) agonist Therapeutics LKWIQEYLEKALN recovery Corp. LY2510924 Lilly Peptide CXCR4 Renal cancer NCT01391130 antagonist SCLC NCT01439568

TG-0054 TaiGen Small molecule Heterocyclic (structure not Multiple myeloma NCT01018979 Biotechnology disclosed publicly) NHL NCT01458288 Patients with Hodgkin NCT00822341 disease and healthy subjects POL6326 Polyphor Ltd. Small molecule Protein epitope mimetics Metastatic breast cancer NCT01837095 technology Hematologic malignancies NCT01413568 Multiple myeloma NCT01105403 Large reperfused ST- NCT01905475 elevation myocardial infarction Healthy volunteers NCT01841476 BKT-140 Biok.Ther. Ltd. Peptide (T140 4F-benzoyl-TN14003 Multiple myeloma NCT01010880 (BL-8040) BioLineRx Ltd. analogue) CML NCT02115672 AML NCT01838395 Mobilization of HSCs to NCT02073019 peripheral blood

NOX-A12 NOXXON Pharma Anti-CXCL12 L-enantiomeric RNA Autologous stem cell NCT00976378 AG oligonucleotide (Spiegelmer) transplantation HSC transplantation NCT01194934 Multiple myeloma NCT01521533 Chronic lymphocytic NCT01486797 leukemia BMS-936564 Bristol-Myers Anti-CXCR4 antibody IgG4 antibody AML (MDX-1338) Squibb Diffuse large B-cell leukemia NCT01120457 Chronic lymphocytic NCT01359657 leukemia Follicular lymphoma Multiple myeloma (Continued on the following page)

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Table 1. CXCR4 antagonists under clinical investigation (Cont'd ) Company/ Amino acid sequences or Trial number/ Name university Description chemical structures Indication reference ALX-0651 Ablynx Anti-CXCR4 Nanobody Healthy volunteers NCT01374503

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

Abbreviations: CML, chronic myeloid leukemia; SCLC, small cell lung cancer. is based on multiple mechanisms: spatial distribution of effec- room for the development of additional agents targeting the tor T cells relatively to tumor, recruitment of tumor-specific CXCR4–CXCL12 axis. CXCR4 inhibition was originally conceived þ T cells from the vessel, and T cells' proliferation activity. In as a strategy to block infection of CD4 T cells by HIV because the PDA model, cancer-associated fibroblasts (CAF) cells may CXCR4 functions as a coreceptor for T-tropic (X4) HIV virus entry regulate the T-cell access to the tumor through release of (20). During its development as an anti-HIV drug, plerixafor CXCL12, which is bound by the PDA cancer cells. Plerixafor, displayed a lack of oral bioavailability and cardiotoxicity. In inhibiting CXCR4, increases accumulation of T cells among addition, its toxicity profile is such that it limits long-term admin- cancer cells, impairing tumor growth and increasing tumor sen- istration, as might be required for metastasis prevention therapy sitivity to anti–PD-L1. The related mechanism must involve (49). Although a 6-month administration was tolerated in WHIM either T cells or myelomonocytic cells, CXCR4-expressing cells. patients, doses used were 4% to 8% of the FDA-approved dose þ Of note, in this model, neither cancer cells nor CAF–FAP cells (26). Finally, plerixafor lacks CXCR4 specificity because it also express CXCR4 (41, 44). CXCR4-dependent modulation of binds the other CXCL12 receptor, CXCR7, as an allosteric agonist immunotherapy was demonstrated in hepatocellular carcino- (50). The ability of CXCR4 antagonists to induce stem cell ma (HCC) models. Treatment of orthotopic HCC with sora- mobilization raised questions about whether the same treatment fenib induced hypoxia responsible for development of resis- might mobilize cells from solid tumors that express CXCR4. In a tance. Sorafenib resistance was mediated by increased presence recent phase I study, the efficacy of the peptidic CXCR4 antagonist þ of immunosuppressive infiltrates (F4/80, tumor-associated LY2510924 was concomitantly evaluated on CD34 mobiliza- macrophages, myeloid-derived suppressive cells, and Tregs) tion versus count of circulating tumor cells (CTC). Although there þ and increased PD-L1 expression in multiple hematopoietic was significant mobilization of CD34 cells upon treatment with cells, including immunosuppressive cells. Because the recruit- LY2510924, no apparent effect on CTC count was observed (51). ment of the immunosuppressive cell population was partially Table 1 lists CXCR4 antagonists in clinical development. The mediated by CXCL12-induced hypoxia, the efficacy of CXCR4 majority of clinical trials have focused on the mobilizing activity inhibition was evaluated. A combined treatment (sorafenib/ of CXCR4 antagonists and were conducted in multiple myeloma, plerixafor/anti–PD-1) showed the most pronounced delay in AML, and chronic lymphatic leukemia (Table 1). Among peptidic tumor growth, probably mediated by increased intratumoral inhibitors, 4F-benzoyl-TN14003 (BKT-140) is a 14-residue bio- penetration and activation of CD8 T lymphocytes in HCC (42). stable synthetic peptide that binds CXCR4 with a greater affinity Moreover, in a mouse model of intraperitoneal papillary compared with plerixafor. Studies in mice demonstrated the epithelial ovarian cancer, CXCR4 antagonism increased tumor efficient and superior mobilization and transplantation of stem cell apoptosis and necrosis, reduced intraperitoneal dissemi- cells collected with G-CSF-BKT140, compared with the single þ nation, and selectively reduced intratumoral FoxP3 Tregs agent alone (52, 53). Additional studies demonstrated anticancer (40). Consistent with these data, plerixafor and the antago- activity (54) and an ability of BKT140 to directly induce cell death nistic CXCR4 peptide R (45, 46) inhibit Treg-suppressive of chronic myeloid leukemia (CML) cells and to revert resistance activity in primary renal cancer specimens in which higher to imatinib mediated by increasing BCL6 (37). A phase I trial numbers of activated Tregs (expressing CTLA-4/CXCR-4/PD-1/ conducted in multiple myeloma patients showed that BKT140 ICOS) were detected. A possible mechanism may involve the was well tolerated and produced a robust mobilization that þ Treg–FOXP3 promoter. resulted in the collection of functional CD34 cells that rapidly Treg–FOXP3 activity is regulated posttranslationally by his- engrafted (55). However, to date, anticancer activity has not been tone/protein acetyltransferases and histone/protein deacetylases demonstrated. (HDAC). Pan-histone/protein deacetylase inhibitors (pan- Evaluation of antimetastatic activity in solid tumors is in HDACi) have been shown to increase FOXP3 acetylation and phase I and II clinical development, and a limited number of DNA binding, enhance Treg production and suppressive activity, results are available. LY2510924 (Eli Lilly and Company) is a and have beneficial effects in the prevention and treatment of potent selective peptide CXCR4 antagonist; a phase I trial show- autoimmune disease and transplant rejection (47). Because ed that its most common drug-related adverse events were CXCR4 signaling transduces also on FOXP3 and HDACis upre- fatigue, injection-site reaction, and nausea. However, its poten- gulated CXCR4 mRNA expression (48), it is possible that CXCR4 tial as an anticancer agent has yet to be established. In the phase I modulation affects the acetylation status of FOXP3 promoter. trial, the best response achieved was stable disease in 9 patients (20%; ref. 51). In two phase II studies, one in renal cell carci- CXCR4 antagonists in development noma (NCT01391130) and one in small cell lung cancer Plerixafor has many positive attributes, including demonstrat- (SCLC; NCT01439568) that evaluated the efficacy of LY2510924 ed antimetastatic potential in preclinical studies; however, there is in combination with sunitinib and carboplatin/etoposide,

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respectively, antitumor efficacy was not achieved. In the SCLC The question at hand is how best to design trials that will trial, there was no improvement in outcome for chemotherapy- circumvent the problem that CXCR4 antagonists may not reduce na€ve patients with extensive-stage disease–small cell lung cancer primary tumor growth in such cancers as ovarian and colorectal (ED-SCLC) when 20 mg of LY2510924 was added to carbo- cancer, where both local and distant metastasis prevention would platin and etoposide (56). This outcome was observed despite be important and where CXCR4 likely plays a role. One proposed preclinical data that were supportive of an antitumor strategy. model of clinical development would be to add it to chemother- The latter included (i) the fact that SCLC cells have been shown apy, such as 5-fluorouracil and oxaliplatin, which are used in to express high levels of CXCR4, and activation of CXCR4 neoadjuvant treatment of locally advanced rectal cancer. This type could induce cell migration and adhesion to stromal cells that of setting would identify a time-limited treatment and a rapid secrete CXCL12, and this in turn could provide growth and drug evaluation of results. Another ideal setting would be for resistance signals to the tumor cells; and (ii) a previous study treatment of glioblastoma following primary surgery, in which that reported CXCR4 antagonists disrupt the CXCR4-mediated it is possible to hypothesize that CXCR4 inhibition coupled to adhesion of SCLC cells to stromal cells, in turn sensitizing radiotherapy may have an additional effect on vasculogenesis SCLC cells to cytotoxic drugs, such as etoposide, by antagonizing ameliorating tumor control, as shown in preclinical studies (61). cell adhesion–mediated drug resistance (39). ED-SCLC may not be an ideal target for development as it often presents with Conclusions extensive primary disease and with metastases at diagnosis, and Extensive preclinical data indicate that targeting the CXCR4– one would expect a CXCR4 antagonist to be most active in primary CXCL12 axis may have several beneficial actions, including (i) stages of metastatic dissemination. affecting CXCR4-expressing primary tumor cells; (ii) synergizing – CTCE-9908, a CXCL12 N-terminus derived peptide that is a with other cancer-targeted therapies; and (iii) modulating the – dimerized sequence of CXCL12 amino acids 1 8, has also under- immune response. Although any or all of these could be valuable gone a phase I and II clinical trial (57). CTCE-9908 was well anticancer strategies, clinical results to date have been disappoint- tolerated, with the most common side effect reported being mild ing. Additional studies and studies with new agents will be to moderate injection site irritation in the 5.0-mg/kg/d dose required to determine whether inhibition of the CXCR4–CXCL12 fi group. However, the magnitude of antitumor ef cacy was at best axis may produce enough anticancer activity to be effective very modest, with 6 patients (30%) with evaluable, although not therapy in the complex setting of human cancer. clinically meaningful, stable disease after one cycle (1 month; ref. 58). Disclosure of Potential Conflicts of Interest Finally, we have recently reported a new family of CXCR4 S. Scala is listed as a co-inventor on a patent, which is co-owned by Istituto antagonists developed through a ligand-based approach Nazionale per lo Studio e la Cura dei Tumori and Consiglio Nazionale delle (45, 46). A three-residue segment identified in CXCL12 that was Ricerche, related to cyclic peptides binding CXCR4 receptor and relative medical fl similar to, but in reverse order, to a peculiar inhibitory chemokine and diagnostic uses. No other potential con icts of interest were disclosed. 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, Acknowledgments The author is deeply indebted to the architect Dr. Barbara Tartarone for help constitutes the core of a cyclic peptide library evaluated for anti- in constructing Fig. 1. CXCR4 activity. Three peptides (R, S, and I) identified as potent CXCR4 antagonists were demonstrated to reduce the develop- Grant Support ment of metastases in in vivo models (45, 46). Recent studies also fi þ S. Scala was supported by the Associazione Italiana per la Ricerca sul Cancro demonstrated that peptides R, S, and I ef ciently mobilize LY6G (AIRC) IG 13192. cells better and longer than AMD3100 and increase bone marrow cellularity, mainly of immature precursors, indicating an active Received April 3, 2015; revised June 9, 2015; accepted June 9, 2015; hematopoietic stimulation. published OnlineFirst July 21, 2015.

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OF8 Clin Cancer Res; 21(19) October 1, 2015 Clinical Cancer Research

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

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