molecular pathways: targeting the cxcr4–cxcl12 axis ... · chemokines act on chemokine receptors...

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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.

Research. on February 23, 2020. © 2015 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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

http://clincancerres.aacrjournals.org/

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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 anti-

cancer therapies.




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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).




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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).




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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




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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




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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




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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




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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




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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




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adverse events were fatigue, injection-site reaction and nausea. However its potential as an anti-

cancer 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).




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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.




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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.

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Grant Support

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

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Table 1. CXCR4 antagonists under clinical investigation

Name Company Description Amino Acid Sequences or Chemical

Structures

Indication

AMD3100 Sanofi Small organic molecule/sc

Hematopoietic stem cell mobilization in patients with non-Hodgkin's lymphoma and multiple myeloma. FDA approved Glioma Acute myeloid leukemia Chronic lymphocytic leukemia Myelokathexis (WHIM syndrome) Ewing's sarcoma Neuroblastoma Brain Tumors

NCT01339039 NCT01141543 NCT01373229 NCT00967785 NCT01288573

AMD 11070 Genzyme Corp.

Small organic molecule

HIV infections HIV infections /X4 tropic virus HIV infections

NCT00089466 NCT00361101 NCT00063804

MSX122 Emory Univ Small organic molecule

Refractory metastatic or locally advanced tumors

NCT005911682

CTCE-9908 antagonist

Chemokine Therapeutics Corp.

Peptide CXCL12-mimetic

KGVSLSYRKRYSLSVGK Solid tumors

(62)

CTCE-0214 agonist

Chemokine Therapeutics Corp.

Peptide CXCL12-mimetic

KPVSLSYRAPFRFF -Linker-

LKWIQEYLEKALN Mobilization BM recovery

(63)

LY2510924 Lilly Peptide CXCR4-

antagonist

Renal cancer SCLC

NCT01391130 NCT01439568

Research.

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TG-0054 TaiGen Biotechnology

Small molecule Heterocyclic (structure not disclosed publicly)

Multiple myeloma Non-hodgkin's lymphoma Hodgkin's disease healthy

NCT01018979NCT01458288 NCT00822341

POL6326 Polyphor Ltd Small molecule Protein Epitope Mimetics (PEM) Technology

Metastatic breast cancerHematologic malignancies Multiple myeloma Large reperfused ST-elevation myocardial infarction Healthy volunteers

NCT01837095NCT01413568 NCT01105403 NCT01905475 NCT01841476

BKT-140 (BL-8040)

Biok.Ther. Ltd BioLineRx Ltd

Peptide (T140 analog) 4F-benzoyl-TN14003

Multiple myelomaChronic myeloid leukemia Acute myeloid leukemia Mobilization of hematopoietic stem cells (HSC) to peripheral blood (PB)

NCT01010880NCT02115672 NCT01838395 NCT02073019

NOX-A12 NOXXON Pharma AG anti-CXCL12 L-enantiomeric RNA oligonucleotide (Spiegelmer)

Autologous stem cell transplantationHematopoietic stem cell transplantation Multiple myeloma Chronic lymphocytic leukemia

NCT00976378NCT01194934 NCT01521533 NCT01486797

BMS-936564 (MDX-1338)

Bristol-Myers Squibb

anti-CXCR4 antibody IgG4 antibody

Acute myelogenous leukemiaDiffuse large B-cell leukemia Chronic lymphocytic leukemia Follicular lymphoma Multiple myeloma

NCT01120457 NCT01359657

ALX-0651 Ablynx anti-CXCR4 Nanobody Healthy volunteers NCT01374503

KRH-1636 Kureha Chemical Industry

Small organic molecule

Anti-HIV-1 activity (64)

Research.

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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:

© 2015 American Association for Cancer Research

CXCR7

CXCL12 CXCL11

CXCR4

CXCR4/7

GRK

ERK1/2

PLC PLC PI3K AC

cAMP

PKA

Lysosome

ERK1/2

RAF

RAS

4EBP1P70S6K

Raptor

mTOR

Cellsurvival,

proliferation

Cellsurvival,

proliferation

CXCL12scavenging

CXCL12scavenging

CXCR4endocytosis,

desensitization

Proliferation,chemotaxis

Proliferation,chemotaxis

Migration,chemotaxis

AKT

PDK1PIP2

IP3

Ca2+

GRK GRK

-arrestin -arrestin -arrestin

CXCR3




Published OnlineFirst July 21, 2015.Clin Cancer Res Stefania Scala Axis-Untapped Potential in the Tumor MicroenvironmentMolecular Pathways: Targeting the CXCR4-CXCL12

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