RU2808667C2 - Cxcr7 inhibitors for cancer treatment - Google Patents

Abstract

FIELD: medicine; oncology.

SUBSTANCE: invention is intended for the treatment of breast cancer in an individual. A method of treating breast cancer in an individual in need thereof comprises administering to the individual of a CXCR7 inhibitor, wherein the individual has aberrant expression of FRS2β and wherein the CXCR7 inhibitor has the structure of Formula I or pharmaceutically acceptable salts thereof or Formula II or pharmaceutically acceptable salts thereof, wherein the radicals are as stated.

EFFECT: use of the invention provides treatment for breast cancer in an individual.

9 cl, 6 dwg, 3 ex

Description

State of the art

Tumor tissues are composed of many heterogeneous cell types; not only tumor cells, but also other cell types, including cancer-associated fibroblasts (CAFs), which are a major component of tumor stroma. Recently, much attention has been paid to the tumor microenvironment as a new therapeutic target, since all these cells appear to support the survival and growth of tumor cells. Accumulating evidence indicates that tumor cells themselves are heterogeneous and include a small number of cancer stem cells (CSCs), which are cancer cells with stemness characteristics, and a large number of rapidly growing differentiated tumor cells. CSCs are thought to control the CSC niche, which is the microenvironment surrounding the CSCs, for their own survival and growth. An environment rich in inflammatory cytokines is thought to be involved in shaping the CSC and tumor microenvironment. Several reports have shown that the transcription factor nuclear factor-κB (NFκB) plays a key role in the production of cytokines, including insulin-like growth factor (IGF) family cytokines and CXC chemokine ligand (CXCL) 12. IGF family cytokines maintain the undifferentiated state of CSCs, and CXCL12 is known to be involved in CAF chemotaxis, and CXCL12 itself activates NFκB. It is known that NFκB is a major inflammatory transcription factor and appears as a heterodimeric complex (RelA and p50 or RelB and p52) that binds to IκB in an inactive state. Ligand stimulation results in phosphorylation of IKKα/β and IκB. Phosphorylated IκB is then ubiquitylated/degraded, and the released NF-κB heterodimer is transported into the nucleus to activate transcription. However, it remains unclear how this occurs early in tumor development, when there are only a few tumor cells in apparently normal tissue.

Breast cancer is the most common type of cancer in women. Recently, much attention has been paid to cancer prevention in order to reduce the number of patients. Emerging evidence suggests that inflammation contributes to breast cancer, but the underlying molecular mechanisms remain unknown. Despite advances in therapeutic strategies, disease-related mortality is still high due to frequent relapses. Accumulating evidence suggests that CSCs represent a major cause of poor prognosis. They are resistant to various stress conditions and are believed to be responsible for tumor initiation, recurrence, and therapeutic resistance. Breast cancer tissues in many cases contain sufficient amounts of stroma, indicating that the CAF-containing tumor microenvironment plays an important role in breast cancer development. Therefore, there is great hope that targeting the tumor microenvironment or CSC niche to eliminate CSCs will be an effective therapeutic strategy in breast cancer. Despite this challenge, reliably effective treatments are still needed.

A subset of breast cancers belong to the human epidermal growth factor receptor 2 (HER2)/ErbB2 subtype, in which cancer cells exhibit amplification of the HER2 gene and/or overexpression of the HER2 protein. Herceptin, a humanized anti-HER2 antibody, is effective against HER2-positive cases; however, Herceptin resistance or relapse still pose significant challenges. Mouse mammary tumor virus (MMTV)-ErbB2 transgenic mice overexpress ErbB2 in mammary tissues, causing tumorigenesis. Breast tissue consists of many branching tubules that end in alveoli, both of which dilate during pregnancy. The epithelium consists of two main cellular layers: luminal cells that surround the inner lumen, and a highly elongated myoepithelium on the other side. Luminal progenitor cells are thought to exist in the luminal cell layer. Evidence suggests that luminal progenitor cells are the cells of origin of mammary tumorigenesis in this model and in human breast cancer. However, effective treatments to prevent or treat the tumorigenesis of these cells remain active areas of study.

ErbB2 homodimerizes or heterodimerizes with other ErbB family members and activates the extracellular signal-regulated protein kinase (ERK) and phosphoinositide 3-kinase (PI3K) signaling pathways, leading to many aspects of tumor biology. ErbB-ERK signaling increases cell proliferation and differentiation depending on the cellular context. ErbB-PI3K signaling activates NFκB.

The adapter protein FRS2β, also called SNT-2 or FRS3, is abundantly expressed in the brain but only in a few regions of other tissues, whereas another member of the FRS2 family, FRS2α, is abundantly expressed in most tissues. Additional information on FRS2β expression is discussed in Gotoh et al. FEBS Lett. 2004. 564 (1-2): 14-8. FRS2β, but not FRS2α, constitutively binds to ErbB family members, including ErbB2, which binds to activated ERK to inhibit feedback and fine-tunes ErbB-ERK signaling. FRS2β also induces subiquitylation and degradation of ErbB1/2. However, the role of FRS2β in vivo, particularly in tumor development, remains unknown.

Taken together, there remains a need in the art to identify the processes that lead to the development of CSC niche environments and agents that can target appropriate agents to modulate, reduce, or prevent tumor development. The present invention satisfies this need and also provides associated advantages.

Brief Disclosure of the Invention

In one aspect, provided herein are methods of treating cancer in an individual in need thereof, the method comprising administering to the individual a CXCR7 inhibitor, wherein the individual has aberrant expression of FRS2β.

In another aspect of the present document, methods are provided for preventing precancerous cells expressing FRS2β from developing into cancer, the method comprising administering a CXCR7 inhibitor to an individual having precancerous cells expressing FRS2β.

In some embodiments, the CXCR7 inhibitor has a structure of Formula I and/or II:

the definitions for each variable group are described in more detail below.

In some embodiments, the CXCR7 inhibitor has the structure of Compound 1

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CXCR7 inhibitor has the structure of compound 2

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CXCR7 inhibitor has the structure of compound 3

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CXCR7 inhibitor has the structure of compound 4

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CXCR7 inhibitor has the structure of compound 5

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CXCR7 inhibitor has the structure of compound 6

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CXCR7 inhibitor has the structure of compound 7

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CXCR7 inhibitor has the structure of compound 8

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CXCR7 inhibitor has the structure of compound 9

or a pharmaceutically acceptable salt thereof.

In some embodiments, the methods presented herein employ one or more therapeutic agents. In some embodiments, one or more therapeutic agents is an IGF1 inhibitor and/or a CXCR4 inhibitor. In some embodiments, the IGF1 inhibitor is an anti-IGF1 antibody.

Other objects, features and advantages of the present invention will become apparent to one skilled in the art from the following detailed description and drawings.

Brief description of drawings

Fig. 1A-H. Deficiency of FRS2β, expressed in luminal cells, strongly delays mammary tumorigenesis. (A) Representative images of β-galactosidase staining for mature female mammary glands of the heterozygous mutant allele of Frs2β. Red arrows indicate FRS2β-positive cells. (B) Diagram of the mammary glands. Many of the branching tubules are surrounded by an inner layer of luminal epithelial cells and an outer layer of myoepithelial epithelial cells. (C) Immunohistological staining of female mammary glands with anti-FRS2β antibody and anti-phosphohistone H3 antibody (top panel) or DAPI (bottom panel). (D) Immunohistological staining of female mammary glands with anti-FRS2β antibody and cytokeratin 18 (upper panels) or cytokeratin 14 (lower panels). (E) Representative NMR imaging of mammary tumors shown in the coronal planes of mice at 14 weeks of follow-up. The left side is the head, the right side is the stomach. (F) Tumor growth in MMTV-neu (+)/Frs2β (+/+) and MMTV-neu (+)/Frs2β (-/-) mice. Tumor sizes were measured once a week for 14 weeks (mean ± SEM, n = 15). FRS2β expression levels were compared by qRT-PCR analysis among virgin, pregnant, and lactating women (mean ± SEM, n = 4, **p < 0.005, *p < 0.01). (G) Representative hematoxylin and eosin (HE)-stained sections of breast tumors. (H) Immunohistochemical staining of Frs2β(+/+) and Frs2β(−/−) mammary tumors using anti-αSMA antibodies. Scale bar: 100 µm.

Fig. 2A-F. FRS2β expressed in luminal progenitor cells supports tumorigenesis derived from tumor cell xenografts. (A) Frs2β(+/+) spherical tumor cells cultured for 14 days, as shown in the representative image, were inoculated into the mammary fat pads of Frs2β(+/+) or Frs2β(−/−) 8-week-old virgins. female mice. (B) Representative tumors were photographed 30 days after transplantation, and (C) the volume of resected tumors was measured. (D) Tumor derived from Frs2β(+/+) spherical tumor cells was observed in Frs2β(+/+) mice but not in Frs2β(−/−) mice (n = 4). The numbers indicate the ratio of the number of tumors to the number of seeded areas. (E) Immunohistochemical staining of MMTV-neu (−) or MMTV-neu (+) female mammary glands with anti-FRS2β antibody and anti-ErbB2 antibody. Arrows indicate FRS2β-positive luminal cells. (F) Mammary epithelial cells were sorted using markers. P1 luminal cell subpopulations (CD49f _low /CD24 _high ) were further sorted using CD61. The P2 luminal progenitor cell subpopulations (CD49f _low /CD24 _high /CD61 ₊ ) were further sorted by FRS2β to generate the P3 subpopulation (CD49f _low /CD24 _high /CD61 ₊ /FRS2β ₊ ).

Fig. 3A-F. Luminal progenitor cells deficient in FRS2β produce fewer cytokines. (A) Representative images of mammospheres derived from Frs2β (+/+) and Frs2β (−/−) mammary epithelial cells cultured in spherical culture medium (SCM). (B) Quantification of sphere formation efficiency of mammosphere cells. N.T., were not treated with cytokine cocktails in SCM. Results are presented as mean ± SEM. n = 4. **p<0.01, *p<0.05. (C) Gene set enrichment analysis (GSEA) was used to compare gene expression profiles of Frs2β (+/+) and Frs2β (−/−) mammosphere cells. Two sets of genes with high activity in Frs2β (+/+) mammosphere cells are shown. (D) Gene set enrichment analysis (GSEA) was used to compare gene expression profiles of Frs2β (+/+) and Frs2β (−/−) precancerous breast epithelial cells. Gene sets with high activity in Frs2β (+/+) or Frs2β (−/−) cells are shown. ES, enrichment score; NES, normalized enrichment score; FDR, false discovery rate. (E) Transcript expression levels of the indicated genes were compared between Frs2β (+/−) and Frs2β (−/−) mammosphere cells using quantitative real-time PCR (qPCR). Results are presented as mean ± SEM. n = 4. **p<0.01. (F) Immunohistochemical staining of Frs2β(+/+) and Frs2β(−/−) mammary tumors using antibodies against αSMA, CXCL12, and IGF1.

Fig. 4A-I. CXCL12 produced from Frs2β(+/+) precancerous breast cells induces tumor spheres and CAF migration. (A) Schematic of coculture of Frs2β(+/+) tumor cells to form spheres in the lower chamber with Frs2β(+/+) precancerous mammary epithelial cells in the upper chamber. (B) Representative images of tumor sphere formation in the presence of Frs2β(+/+) mammary epithelial cells treated with control IgG (400 nM) or IGF1 neutralizing antibody (Nab) (400 nM). N.T., without treatment (without co-culture with mammary epithelial cells). Scale bar: 100 µm. (C) Quantification of tumor sphere formation efficiency. Results are shown as means ± SEM. n = 4. ***p<0.001, **p<0.01. (D) Schematic of coculture of Frs2β (+/+) or Frs2β (−/−) mammary cells in the lower chamber with Frs2β (+/+) or Frs2β (−/−) CAFs in the upper chamber. (E) Cxcl12 expression levels were compared between Frs2β (+/+) or Frs2β (−/−) mammary cells in the upper chamber by qPCR. Results are presented as mean ± SEM. n = 6. ***p<0.001. (F) Representative images of migrated Frs2β(+/+) CAFs cocultured with Frs2β(+/+) or Frs2β(−/−) mammary cells in the upper chamber for 24 hours. (G) Quantification of migrated Frs2β(+/+) CAFs. Results are presented as mean ± SEM. n = 4. **p<0.01. (H) Representative images of migrated CAFs co-cultured with Frs2β(+/+) cancer cells for 24 hours. Cells were treated with compound 1 at the indicated concentration and/or + 0.1 mg/ml AMD3100) or served as a control. (I) Quantification of migrated CAFs co-cultured with Frs2β(+/+) cancer cells for 24 hours. Results are presented as mean ± SEM.n = 4. ***p<0.001 and **p<0.01.

Fig. 5A-K FRS2β-dependent increase in AKT-NFkB activation increases IGF1 and CXCL12 production, promoting tumorigenesis. (A) Schematic of DHMEQ treatment of cultured Frs2β(+/+) precancerous mammary epithelial cells in vitro. (B) Expression levels of Igf1, Cxcl12, and IκBα in Frs2β(+/+) precancerous mammary epithelial cells treated with the indicated concentration of DHMEQ were compared by qPCR. Results are shown as means ± SEM. n = 4. **p<0.01. (C) Immunoblot analysis of the indicated proteins in lysates of Frs2β (+/+) or Frs2β (−/−) precancerous breast tissues. Actin was used as a loading control. (D) Immunoblot analysis of cytoplasmic and nuclear expression levels of the indicated proteins (control as nuclear protein) in lysates of Frs2β (+/+) or Frs2β (−/−) mammary tissues. PARP1 was used as a representative protein in the nucleus. Actin was used as a loading control. (E) Immunoblot analysis of the indicated proteins in Frs2β (+/+) or Frs2β (−/−) mammary tissue lysates. Actin was used as a loading control. (F) Schematic of DHMEQ treatment of Frs2β(+/+) mice in vivo. Mice were intraperitoneally administered 10 μg/g DHMEQ once daily for 3 weeks. (G) Immunohistochemical staining of Frs2β (+/+) mammary tissues with or without 3 weeks of treatment with 10 μg/g DHMEQ or Frs2β (−/−) mammary tissues using anti-RelA antibodies. Scale bar: 50 µm. (H) Expression levels of Igf1 and Cxcl12 in Frs2β (+/+) mammary tissues with or without 3 weeks of 10 μg/g DHMEQ treatment. N.T., without processing. Results are shown as means ± SEM. n = 4. ***p<0.001 and **p<0.01. (I) Treatment of mice with CXCR7 inhibitor and/or IGF1 antibody reduces tumor volume. Frs2β(+/+) tumor sphere cells were inoculated into the mammary fat pads of 8-week-old virgin female MMTV-neu(+)/Frs2β(+/+) mice. After 7 days, mice were intraperitoneally injected with 0.1 μg/g IGF1 antibody (R&D Biosystems) once a week and/or 1 μg/g AMD3100 (Sigma) once a day and 1.5 μg/g compound 1 once a day. Representative tumors were photographed at day 35 post-transplantation of CXCL12 Inh, a combination of AMD3100 and CCX771. Volumes (J) and weights (K) of tumors were measured in mice treated as in (I). Results are presented as mean ± SEM, nK = 4, *p < 0.05.

Fig. 6 A-G Tumor cells expressing FRS2β produce IGF1 and CXCL12 and are associated with abundant stroma and poor prognosis (A) Immunohistochemical staining of Frs2β (+/+) breast tumors with anti-FRS2b and anti-ErbB2 antibodies. Scale bar, 25 µm. (B) Expression levels of Cxcl12 and Igf1 were compared between Frs2β (+/+) and Frs2β (−/−) tumor cells by qPCR. Results are shown as means ± SEM. n = 4. ***p< 0.001. (C) Immunohistochemical staining with antibodies against IGF1 and CXCL12. Scale bar, 200 μm (D) Tissue panels were subjected to immunohistochemical staining with anti-FRS2b antibody or Masson's trichrome staining to detect collagen in the stroma. Arrows indicate the stromal region. Scale bar: 50 µm. (E) Tumor samples were divided into three groups according to the ratio of tumor stromal area to total tumor area (+: 0-10%, ++; 10-20%, +++; >20%). The median of FRS2b staining levels was used for cutoff values. n = 30. (F) Kaplan-Meier survival curve using the Uppsala cohort (GSE3494). Medians were used for thresholding. The P-value was obtained using the log-rank test. (G) FRS2b can trigger cytokine production in a subset of luminal cells, resulting in the creation of a cytokine-rich precancerous microenvironment (top left panel). Once CSCs emerge in the precancerous microenvironment, they acquire the ability to self-renew in the presence of IGF1 and produce tumor cells via CXCL12-mobilized stromal cells, which subsequently develop into CAFs. CSCs and tumor cells can independently produce IGF1 and CXCL12, leading to rapid growth and tumorigenesis (lower left panel). Without FRS2β, cytokines remain at low levels and the corresponding precancerous microenvironment is not created (top right panel); even when CSCs emerge, they fail to grow efficiently (lower right panel).

Carrying out the invention

I. General

The present description demonstrates that aberrant expression of FRS2β maintains suitable microenvironmental conditions for tumor growth and plays a critical role in establishing a cytokine-rich CSC niche. Surprisingly, the deleterious effects of this expression can be effectively modulated by administering a CXCR7 inhibitor or a CXCR7 inhibitor in combination with another therapeutic agent.

II. Definitions

The term “alkyl,” by itself or as part of another substituent, means, unless otherwise indicated, a straight or branched chain hydrocarbon radical having the indicated number of carbon atoms (ie, C _1-8 means one to eight carbon atoms). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like. The term "alkenyl" refers to an unsaturated alkyl group having one or more double bonds. Likewise, the term "alkynyl" refers to an unsaturated alkyl group having one or more triple bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3 -butynyl, as well as higher homologues and isomers. The term "cycloalkyl" refers to hydrocarbon rings having a specified number of ring atoms (eg, C _3-6 cycloalkyl) and being fully saturated or having no more than one double bond between the vertices of the ring. "Cycloalkyl" also means bicyclic and polycyclic hydrocarbon rings, such as, for example, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, etc. The term "cycloalkenyl" refers to a cycloalkyl group having at least one double bond between the vertices of the ring. Cyclopentenyl and cyclohexenyl are examples of cycloalkenyl. The term "spirocycloalkyl" refers to a cycloalkyl group in which one tip of the ring is attached to two other non-hydrogen parts of the molecule. A spirocycloalkyl substituent is a substituent in which two carbon atoms of the alkylene chain (usually the ends of the alkylene chain) are attached to the same carbon atom in the rest of the molecule. The term “heterocycloalkyl” refers to a cycloalkyl group that contains from one to five heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom(s) are optionally quaternized. Heterocycloalkyl may be a monocyclic, bicyclic or polycyclic ring system. Non-limiting examples of heterocycloalkyl groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine , pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine and the like. A heterocycloalkyl group can be attached to the remainder of the molecule through a ring carbon or a heteroatom.

The term "alkylene" by itself or as part of another substituent means a divalent radical derived from an alkane, as illustrated by the following formula -CH ₂ CH ₂ CH ₂ CH ₂ -. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with groups containing 10 or fewer carbon atoms being preferred groups in the present invention. "Lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, typically having four or fewer carbon atoms. Likewise, "alkenylene" and "alkynylene" refer to unsaturated forms of "alkylene" having double or triple bonds, respectively.

As used herein, a wavy line, " ", which crosses a single, double, or triple bond in any chemical structure depicted herein, represents the point addition of a single, double, or triple bond to the rest of the molecule.

The terms "alkoxy group", "alkylamino group" and "alkylthio group" (or thioalkoxy group) are used in their conventional sense and refer to those alkyl groups that are attached to the rest of the molecule through an oxygen atom, an amino group or a sulfur atom, respectively. Additionally, for dialkylamino groups, the alkyl moieties may be the same or different, and may also be combined to form a 3-7 membered ring with a nitrogen atom to which each is attached. Accordingly, the group represented by NR ^a R ^b includes piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl and the like.

The terms "halo" or "halogen" by themselves or as part of another substituent mean, unless otherwise specified, a fluorine, chlorine, bromine or iodine atom. In addition, terms such as "haloalkyl" include monohaloalkyl and polyhaloalkyl. For example, the term "C _1-4 haloalkyl" means trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl and the like.

The term "aryl" means, unless otherwise specified, a polyunsaturated, usually aromatic hydrocarbon group, which may be a single ring or multiple rings (up to three rings) that are fused together or linked covalently. The term “heteroaryl” refers to aryl groups (or rings) that contain from one to five heteroatoms selected from N, O and S, where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom(s) are optionally quaternized. The heteroaryl group can be attached to the rest of the molecule via a heteroatom. Non-limiting examples of aryl groups include phenyl, naphthyl and biphenyl, while non-limiting examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimindinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzenepyrazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotri azolyl, benzisoxazolyl, isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridines, benzothiaxolyl, benzofuranyl, benzothienyl, indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, tria zolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, thienyl and the like. The substituents for each of the above aryl and heteroaryl ring systems are selected from the group of suitable substituents described below.

The term "arylalkyl" includes those radicals in which an aryl group is attached to an alkyl group (eg, benzyl, phenethyl, and the like). Likewise, the term “heteroarylalkyl” is intended to include those radicals in which a heteroaryl group is attached to an alkyl group (eg, pyridylmethyl, thiazolylethyl, and the like).

The above terms (eg, "alkyl", "aryl" and "heteroaryl") will in some embodiments include both substituted and unsubstituted forms of the specified radical. The following are the preferred substituents for each type of radical.

Substituents for alkyl radicals (including groups often called alkylene, alkenyl, alkynyl and cycloalkyl) can be various groups selected from: -halogen, -OR', -NR'R”, -SR', -SiR'R”R” ', -OC(O)R', -C(O)R', -CO ₂ R', -CONR'R”, -OC(O)NR'R”, -NR”C(O)R', -NR'-C(O)NR”R”', -NR”C(O) ₂ R', -NH-C(NH ₂ )=NH, -NR'C(NH ₂ )=NH, -NH- C(NH ₂ )=NR', -S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R”, -NR'S(O) ₂ R”, -CN and - NO ₂ in the range from zero to (2 m'+1), where m' represents the total number of carbon atoms in such a radical. R', R” and R'” are each independently hydrogen, unsubstituted C _1-8 alkyl, unsubstituted aryl, aryl substituted with 1-3 halogen atoms, unsubstituted C _1-8 alkyl, C _1-8 alkoxy or C _1-8 thioalkoxy groups, or unsubstituted aryl-C _1-4 alkyl groups. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, -NR'R'' is intended to include 1-pyrrolidinyl and 4-morpholinyl.

Likewise, the substituents for aryl and heteroaryl groups vary and are usually selected from: -halogen, -OR', -OC(O)R', -NR'R”, -SR', -R', -CN, _-NO2 , -CO ₂ R', -CONR'R”, -C(O)R', -OC(O)NR'R”, -NR”C(O)R', -NR”C(O) ₂ R' , -NR'-C(O)NR”R”', -NH-C(NH ₂ )=NH, -NR'C(NH ₂ )=NH, -NH-C(NH ₂ )=NR', - S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R”, -NR'S(O) ₂ R”, -N ₃ , perfluoro(C ₁ -C ₄ )alkoxy and perfluoro(C ₁ -C ₄ )alkyl in an amount from zero to the total number of open valencies in the aromatic ring system; and wherein R', R” and R”' are independently selected from hydrogen, C _1-8 alkyl, C _1-8 haloalkyl, C _3-6 cycloalkyl, C _2-8 alkenyl, C _2-8 alkynyl, unsubstituted aryl and heteroaryl ( unsubstituted aryl)-C _1-4 alkyl and unsubstituted aryloxy -C _1-4 alkyl. Other suitable substituents include each of the above aryl substituents attached to a ring atom by an alkylene bond of 1 to 4 carbon atoms.

Two substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be replaced by a substituent of the formula -TC(O)-(CH ₂ ) _q -U-, in which T and U are independently -NH-, -O-, -CH ₂ - or a single bond, and q is an integer from 0 to 2. Alternatively, two substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be replaced by a substituent of the formula -A-(CH ₂ ) _r -B-, where A and B independently represent represents -CH ₂ -, -O-, -NH-, -S-, -S(O)-, -S(O) ₂ -, -S(O) ₂ NR'- or a single bond, and r represents an integer from 1 to 3. One of the single bonds of the new ring thus formed may optionally be replaced by a double bond. Alternatively, two substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be replaced by a substituent of the formula -(CH ₂ ) _s -X-(CH ₂ ) _t -, in which s and t are integers from 0 to 3 independently of each other and X is -O-, -NR'-, -S-, -S(O)-, -S(O) ₂ - or -S(O) ₂ NR'-. The R' substituent in -NR'- and -S(O) ₂ NR'- is selected from hydrogen or unsubstituted C ₁ - ₆ alkyl.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

As used herein, the terms “progenitor cells” and “stem cells” are used interchangeably. “Progenitor cells” and “stem cells” refer to cells that, in response to certain stimuli, can form differentiated cell lineages, including, but not limited to, hematopoietic, mesenchymal, epithelial, neuronal, renal or myeloid cells. The presence of progenitor/stem cells can be assessed by the ability of the cells in the sample to form various types of colony-forming units, including, for example, CFU-GM (colony-forming units, granulocyte-macrophage); CFU-GEMM(colony forming units, polyvalent); BFU-E(burst-forming units, erythroid); HPP-CFC (colony-forming cells with high proliferative potential); or other types of differentiated colonies that can be obtained in culture using known protocols. Hematopoietic progenitor/stem cells are often positive for CD34. However, some stem cells do not contain this marker. These CD34+ cells can be analyzed using fluorescence activated cell sorting (FACS), and thus their presence in the sample can be assessed using this method. Alternatively, such cells can be analyzed by FACS for the presence of c-kit receptor (CD117), absence of lineage-specific markers (e.g. CD2, CD3, CD4, CD5, CD8, NK1.1, B220, TER-119 and Gr -1 in mice and CD3, CD14, CD16, CD19, CD20 and CD56 in humans).

The term "pharmaceutically acceptable salts" includes salts of active compounds that are prepared with relatively non-toxic acids or bases, depending on the specific substituents found in the compounds described herein. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be prepared by reacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of salts derived from pharmaceutically acceptable inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganese, manganese, potassium, sodium, zinc and the like. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally occurring amines and the like, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, piperindine, piperazine, piperidine , triethylamine, trimethylamine, tripropylamine, tromethamine and the like. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be prepared by reacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrocarboxylic, phosphoric, monohydrophosphoric, dihydrophosphoric, sulfuric, monohydrosulfuric, hydroiodic or phosphorous acids and the like, as well as salts , obtained from relatively non-toxic organic acids, such as acetic, propionic, isobutyric, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, etc. Also included are salts of amino acids, such as arginate and the like, and salts of organic acids, such as glucuronic or galactunic acids and the like (see, for example, Berge, S. M., et al, “Pharmaceutical Salts,” Journal of Pharmaceutical Science, 1977, 66, 1-19). Some specific compounds of the present invention contain both basic and acidic functional groups, which allow the compounds to be converted into either base or acid addition salts.

Neutral forms of the compounds can be regenerated by reacting the salt with a base or acid and isolating the parent compound in the usual manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility, in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for purposes of the present invention.

In addition to salt forms, the present invention provides compounds that are in prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to produce the compounds of the present invention. In addition, prodrugs can be converted into the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.

Some of the compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, solvated forms are equivalent to non-solvated forms and are intended to be included within the scope of the present invention. Some of the compounds of the present invention may exist in a variety of crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

Some compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds; Racemates, diastereomers, geometric isomers, regioisomers, and individual isomers (eg, individual enantiomers) are intended to be included within the scope of the present invention. In some embodiments, the compounds of the invention are present in enantiomerically enriched form, where the amount of enantiomeric excess for a particular enantiomer is calculated by known methods. Preparation of enantiomerically enriched forms is also well known in the art and can be accomplished, for example, using chiral resolution by chromatography or chiral salt formation. In addition, the present invention provides for various conformers as well as various rotamers. Conformers are conformational isomers that can differ in rotations around one or more σ bonds. Rotamers are conformers that differ only in their rotation around a single σ bond. In addition, the compounds of the present invention may also contain unusual proportions of atomic isotopes at one or more of the atoms that make up such compounds. Accordingly, in some embodiments, the compounds of the invention are present in an isotopically enriched form. Unnatural proportions of an isotope can be defined as ranging from the amount found in nature to the amount consisting of 100% of the atom in question. For example, the compounds may include radioactive isotopes such as tritium ( ^3H ), iodine-125 ( ^125I ) or carbon-14 ( ^14C ), or non-radioactive isotopes such as deuterium ( ^2H ) or carbon-13 ( ¹³ C). Such isotopic variations may provide additional benefits over those described elsewhere in this application. For example, isotopic variants of the compounds of the present invention may find additional utility, including, but not limited to, as diagnostic and/or imaging reagents or as cytotoxic/radiotoxic therapeutics. In addition, isotopic variants of the compounds of the invention may have altered pharmacokinetic and pharmacodynamic characteristics that may enhance safety, tolerability, or efficacy during treatment. All isotopic variants of the compounds of the present invention, radioactive or not, are intended to be included within the scope of the present invention.

"CXCR7", also referred to as "RDC1" or "CCXCKR2", refers to a seven-transmembrane domain putative G protein-coupled receptor (GPCR). The canine ortholog of CXCR7 was originally identified in 1991, see Libert et al. Science244:569-572 (1989). The dog sequence is described in Libert et al., Nuc. Acids Res 18(7):1917 (1990). The mouse sequence is described, for example, in Heesen et al., Immunogenetics 47:364-370 (1998). The human sequence is described, for example, in Sreedharan et al., Proc. Natl. Acad. Sci. USA 88:4986-4990 (1991), which erroneously described the protein as a receptor for vasoactive intestinal peptide.

The term “therapeutically effective amount” means the amount of a subject compound that will produce a biological or medical response in a cell, tissue, system, or animal, such as a human, that is desired by a researcher, veterinarian, physician, or other treatment provider.

The term “composition” as used herein is intended to cover a product containing the specified ingredients in the specified amounts, as well as any product that is directly or indirectly the result of a combination of the specified ingredients in the specified amounts. By "pharmaceutically acceptable" is meant that the carrier, diluent or excipient must be compatible with the other ingredients of the drug and not harmful to its recipient.

III. Detailed description of embodiments

A. Methods

In one aspect, methods are provided herein for treating cancer in an individual in need thereof, the method comprising administering to the individual a CXCR7 inhibitor, wherein the individual exhibits aberrant expression of FRS2β.

In another aspect, methods are provided herein for preventing precancerous cells expressing FRS2β from developing into cancer, the method comprising administering to an individual having precancerous cells expressing FRS2β a CXCR7 inhibitor.

As described in the Background section, FRS2β is abundantly expressed in the brain, but only in a few regions in other tissues. Thus, many tissues do not naturally express FRS2β. As demonstrated herein, aberrant expression of FRS2β in cells that do not otherwise express this protein can provide a CSC niche and lead to tumorigenesis.

It is understood that aberrant expression refers to the expression of a protein in a cell, tissue, organ or body fluid of a subject that does not normally produce the protein in a healthy individual (inappropriate expression) or the expression of higher levels of protein in a cell, tissue, organ or body fluid of a subject. than is found in the same type of cell, tissue, organ, or body fluid of a healthy individual (differential expression). In some embodiments, aberrant expression of FRS2β is at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40% at least about 45%, at least about 50%, or greater FRS2β expression than in a healthy person. One skilled in the art will appreciate that FRS2β expression can be determined using methods known in the art. In some embodiments, FRS2β expression can be detected as described in the disclosed methods. In some embodiments, FRS2β expression can be detected using immunohistochemistry. In various embodiments, aberrant expression is detected using an ELISA assay.

Many CXCR7 inhibitors are known in the art, and further details of possible CXCR7 inhibitors useful in the present disclosure are further discussed in the sections below.

A preferred method of treating cancer involves administering a therapeutically effective amount of one or more of the previously mentioned compounds (or salts thereof) to a cancer patient for a time sufficient to treat the cancer.

In addition to primates such as humans, a variety of other mammals can be treated according to the method of the present invention. For example, mammals may be treated, including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other types of cattle, sheep, horses, dogs, cats, rodents or mice. However, this method can also be practiced in other species, such as bird species (eg chickens).

In some cases, CXCR7 inhibitors are administered to treat cancers, for example, carcinomas, gliomas, mesotheliomas, melanomas, lymphomas, leukemias (including acute lymphocytic leukemias), adenocarcinomas, breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, lymphoma, prostate cancer and Burkitt's lymphoma, colon cancer, colorectal cancer, esophageal cancer, stomach cancer, pancreatic cancer, such as liver and biliary tract cancer, gallbladder cancer, small intestine cancer, rectal cancer, kidney cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, cervical cancer, vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenal cancer, endocrine pancreatic cancer, carcinoid cancer, bone cancer, skin cancer , retinoblastoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (see Cancer: Principles and Practice (DeVita, V.T. et al. eds 1997) for additional cancers).

In some embodiments, the cancer being treated herein is breast cancer.

In some embodiments, the individual has been diagnosed with aberrant expression of FRS2β prior to administration of a CXCR7 inhibitor or additional therapeutic agent.

B. CXCR7 inhibitors

In some embodiments, CXCR7 inhibitors have a Formula I structure

or pharmaceutically acceptable salts, hydrates, N-oxides, isotopically enriched or enantiomerically enriched versions thereof, where

index n is an integer from 0 to 2;

each R ¹ , if present, is independently selected from the group consisting of C _1-4 alkyl, -CO ₂ ^Ra , -X-CO ₂ ^Ra , -CONR ^a R ^b and -X-CONR ^a R ^b ;

R ² and R ³ are each a member that is independently selected from the group consisting of H, -R ^a , -XR ^a , -XNR ^a R ^b , -XNHCONR ^a R ^b , -XNHCOR ^a , -XO-CONR ^a R ^b , -XNHSO ₂ R ^a , -CO ₂ R ^a , -X-CO ₂ R ^a , -CONR ^a R ^b and -X-CONR ^a R ^b ; or taken together represent an oxo group;

C ¹ is selected from the group consisting of monocyclic or fused bicyclic aryl and heteroaryl, in which the heteroaryl group has 1-3 heteroatoms as ring members selected from N, O and S; and wherein said aryl and heteroaryl groups are optionally substituted with 1 to 3 R ⁴ substituents;

C ² is a monocyclic four-, five-, six- or seven-membered ring selected from the group consisting of benzene, heteroaromatic compound, cycloalkane and heterocycloalkane, where the heteroaromatic and heterocycloalkane rings contain from 1 to 3 heteroatoms as ring members selected from N, O and S; and wherein each of said monocyclic C ² rings is optionally substituted with 1 to 3 R ⁵ substituents;

C ³ is selected from the group consisting of hydrogen, C _1-8 alkyl, C _3-8 cycloalkyl, aryl, aryl-C _1-4 alkyl, heteroaryl, heteroaryl-C _1-4 alkyl and four to six membered heterocycloalkyl, in wherein the heterocycloalkyl group or heterocycloalkyl moiety contains from 1 to 3 heteroatoms selected from N, O and S, and wherein the heteroaryl group contains from 1 to 3 heteroatoms as ring members selected from N, O and S, and each C ³ is optionally substituted one of 1-3 substituents R ⁶ ;

each R ⁴ is independently selected from the group consisting of halogen, -CN, -NO ₂ , -R ^c , -CO ₂ R ^a , -NR ^a R ^b , -OR ^a , -X-CO ₂ R ^a , -CONR ^a R ^b and -X-CONR ^a R ^b ;

wherein each of R ¹ , R ² , R ³ and R ⁴ , each R ^a and R ^b are independently selected from hydrogen, C _1-8 alkyl, C _3-7 cycloalkyl, C _1-8 haloalkyl, and four to six membered heterocycloalkyl, or when attached to the same nitrogen atom can be combined with a nitrogen atom to form a four-, five-, or six-membered ring having from 0 to 2 additional heteroatoms as ring members selected from N, O or S; within R ⁴ , each R ^c is independently selected from the group consisting of C _1-8 alkyl, C _1-8 haloalkyl, C _3-6 cycloalkyl, aryl and heteroaryl, and wherein the aliphatic and cyclic moieties of R ^a , R ^b and R ^c are optional additionally substituted with one to three halogen, hydroxy, methyl, alkoxy, amino, alkylamino, dialkylamino, carboxamide, carboxyalkyl ether, carboxylic acid, heteroaryl, and four to six-membered heterocycloalkyl groups; and wherein the heterocycloalkyl portions of R ² , R ³ and R ⁴ are optionally substituted with an oxo group; and, optionally, when two R ⁴ substituents are on adjacent atoms, combine to form a fused five- or six-membered ring, with carbon and oxygen atoms as ring members;

each R ⁵ is independently selected from the group consisting of halogen, -CN, -NO ₂ , -R ^f , -CO ₂ R ^d , -COR ^d , -NR ^d R ^e , -OR ^d , -X-CO ₂ R ^d , -CONR ^d R ^e and -X-CONR ^d R ^e ; wherein each R ^d and R ^e are independently selected from hydrogen, C _1-8 alkyl, C _1-8 haloalkyl, C _3-6 cycloalkyl, C _3-6 cycloalkylalkyl and four to six membered heterocycloalkyl or when attached to the same atom nitrogen can be combined with a nitrogen atom to form a five- or six-membered ring having from 0 to 2 additional heteroatoms as ring members selected from N, O or S; each R ^f is independently selected from the group consisting of C _1-8 alkyl, C _1-8 haloalkyl and C _3-6 cycloalkyl, and in which the aliphatic and cyclic moieties R ^d , R ^e and R ^f are optionally further substituted from one to three halogen, hydroxy, methyl, alkoxy, amino, alkylamino, dialkylamino, carboxamide, carboxyalkyl ether, carboxylic acid, heteroaryl, four to six membered heterocycloalkyl groups;

each R ⁶ is independently selected from the group consisting of halogen, -CN, -NO ₂ , -R ⁱ , -CO ₂ R ^g , -COR ^g , -NR ^g R ^h , -OR ^g , -X-CO ₂ R ^g , -X-COR ^g , -CONR ^g R ^h and -X-CONR ^g R ^h , wherein each R ^g and R ^h are independently selected from hydrogen, C _1-8 alkyl and C _1-8 haloalkyl; each R ⁱ is independently selected from the group consisting of C _1-8 alkyl and C _1-8 haloalkyl; And

each X represents a linker group having the formula -(CH ₂ ) _m O(CH ₂ ) _p - in which the subscripts m and p are integers from 0 to 5, and m + p is from 0 to 6, where the methylene groups are optionally substituted with one or two methyl groups.

In some embodiments, the CXCR7 inhibitor has the structure of Compound 1

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CXCR7 inhibitor has the structure of compound 2

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CXCR7 inhibitor has the structure of compound 3

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CXCR7 inhibitor has the structure of compound 4

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CXCR7 inhibitor has the structure of compound 5

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CXCR7 inhibitor has the structure of compound 6

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CXCR7 inhibitor is selected from the compounds or pharmaceutical compositions disclosed in PCT Publication No. WO2010/054006 arising from PCT Application No. US2009/063298 filed November 4, 2009 by ChemoCentryx, the contents of which are incorporated herein for all purposes.

In some embodiments, CXCR7 inhibitors have a Formula II structure

or a pharmaceutically acceptable salt, hydrate, N-oxide, isotopically enriched or enantiomerically enriched version or rotamer thereof, where

each of the ring vertices X ^a , X ^b and X ^c are independently selected from the group consisting of N, NH, N(R ² ), O, CH and C(R ² );

index n is 0, 1 or 2;

Z is selected from the group consisting of

(i) monocyclic or fused bicyclic aryl and heteroaryl, in which the heteroaryl group has from 1 to 4 heteroatoms as ring members selected from N, O and S; and wherein said aryl and heteroaryl groups are optionally substituted with 1 to 5 R ⁵ substituents;

(ii) a monocyclic four-, five-, six- or seven-membered ring, which is selected from the group consisting of cycloalkane and heterocycloalkane, in which the heterocycloalkane rings have from 1 to 3 heteroatoms as ring members selected from N, O and S; and wherein each of said monocyclic Z rings is optionally substituted with 1 to 3 R ⁵ substituents;

R ¹ is a member selected from the group consisting of H and C _1-8 alkyl, in which the alkyl moiety is optionally substituted with halogen, -NR ^a R ^b , -OR ^a , -CO ₂ R ^a , and -CONR ^a R ^b ;

each R ² is independently selected from the group consisting of H, halogen, CN, C _1-8 alkyl, C _1-8 haloalkyl, C _1-8 hydroxyalkyl, -OR ^a , -CO ₂ R ^a , -X-CO ₂ R ^a , -NR ^a R ^b , -CONR ^a R ^b and -X-CONR ^a R ^b ;

R ³ is a member selected from the group consisting of H, C _1-8 alkyl, C _1-8 haloalkyl, C _1-8 hydroxyalkyl, -CO ₂ ^Ra , -X-CO ₂ ^Ra , -CONR ^a R ^b and -X-CONR ^a R ^b ;

each R ⁴ , if present, is a member independently selected from the group consisting of C _1-8 alkyl, C _1-8 haloalkyl, C _1-8 hydroxyalkyl, -OR ^a , -CO ₂ ^Ra , -X-CO ₂ R ^a , -NR ^a R ^b , -CONR ^a R ^b and -X-CONR ^a R ^b ;

each R ⁵ is a member independently selected from the group consisting of halogen, CN, -X-CN, C _1-8 alkyl, C _3-8 cycloalkyl, C _3-8 cycloalkenyl, C _3-5 spirocycloalkyl, C _{2- 8} alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _1-8 hydroxyalkyl, -OR ^a , -CO ₂ R ^a , -X-CO ₂ R ^a , -NR ^a R ^b , -CONR ^a R ^b , -X-CONR ^a R ^b , aryl, 5- or 6-membered heteroaryl and 3-, 4-, 5- or 6-membered heterocyclyl, in which the heteroatoms present as ring vertices of the heteroaryl and heterocyclic rings are selected from N, O and S, and in which the aryl, heteroaryl and heterocyclic parts of R ⁵ are optionally further substituted with 1-3 R ^a ;

each R ^a and R ^b are independently selected from the group consisting of hydrogen, hydroxyl, halogen, cyano, C _1-8 alkyl, C _1-8 alkoxy, C _1-8 haloalkyl, C _3-6 cycloalkyl, C _3-6 cycloalkylalkyl , amino groups, C _1-8 alkylamino groups, diC _1-8 alkylamino groups, carboxamide, carboxy-C _1-4 alkyl ester, carboxylic acid and -SO ₂ -C _1-8 alkyl;

each X represents a C _1-4 alkylene linker group or a linker group having the formula - (CH ₂ ) _m O(CH ₂ ) _p - where the subscripts m and p are integers from 0 to 5, and m + p is from 0 to 6, in which any of the methylene moieties of X is optionally substituted with one or two methyl groups.

In some embodiments, the CXCR7 inhibitor has the structure of compound 7

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CXCR7 inhibitor has the structure of compound 8

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CXCR7 inhibitor has the structure of compound 9

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CXCR7 inhibitor is selected from the compounds or pharmaceutical compositions disclosed in PCT Publication No. WO2014/085490 resulting from PCT Application No. US2013/072067 filed November 26, 2013 by ChemoCentryx, the contents of which are incorporated herein for all purposes.

C. Combination therapy

Methods of treating cancer disclosed herein may further include one or more additional therapeutic agents.

Additional therapeutic agents that can be used in the present disclosure include compounds or compositions that have anticancer activity. In some embodiments, the CXCR7 modulators of the present invention can be administered in combination with chemotherapeutic agents or radiation.

Additional examples of other therapeutic agents that can be combined with a compound or composition of the present invention, administered alone or in the same pharmaceutical compositions, include, but are not limited to: IGF1 inhibitor (e.g., antibody or small molecule), CXCR4 inhibitor (e.g., AMD3100 ), immunomodulatory agent, cisplatin, paclitaxel, methotrexate, cyclophosphamide, ifosfamide, chlorambucil, carmustine, carboplatin, vincristine, vinblastine, thiotepa, lomustine, semustine, 5-fluorouracil and cytarabine. In some embodiments, one or more additional therapeutic agents may be an anti-IGF1 antibody and/or a CXCR4 inhibitor. In some embodiments, one or more additional therapeutic agents is a CXCR4 inhibitor. In some cases, one or more additional therapeutic agents is an anti-IGF1 antibody.

A number of CXCR4 inhibitors are known in the art, including small molecules, peptides and antibodies, all of which are useful in the present disclosure. Some exemplary CXCR4 inhibitors include AMD3100, as well as the CXCR4 inhibitors presented in WO2007115232, WO2007115231, US20070275965, US20130289020, US20140286936 and US20170226106, the contents of each of which are incorporated herein for all purposes.

A number of small molecule inhibitors and CXCR4-like antibodies targeting IGF1 are known. Examples of inhibitors include: AG538, AG1024, NVP-AEW541 and figitumumab, as well as inhibitors presented in US20090068110, US20140045832, US20050281812, US20050244408, US20120005767, US20140044720 and US2 0080161278, the contents of each of which are incorporated herein for all purposes.

The weight ratio of the compound of the present invention to the second active ingredient may vary and will depend on the effective dose of each ingredient. Typically an effective dose of each will be used. Thus, for example, when a compound of the present invention is combined with a second anticancer agent, the weight ratio of the compound of the present invention to the second agent will generally be in the range of from about 1000:1 to about 1:1000, preferably from about 200:1 to about 1:200. Combinations of a compound of the present invention and other active ingredients will generally also fall within the above-mentioned range, but an effective dose of each active ingredient should be used in each case.

It is understood that such administration may occur before, after, or simultaneously with the second therapeutic agent, such that the therapeutic effects of the second agent are enhanced relative to administration of the second agent in the absence of the CXCR7 modulator. The selection of suitable agents for use in combination therapy can be made by one skilled in the art in accordance with generally accepted pharmaceutical principles. A combination of therapeutic agents can act synergistically, and by using this approach, therapeutic efficacy can be achieved with lower dosages of each agent, thereby reducing the likelihood of adverse side effects.

D. Methods of administration

In general, the methods of treatment provided herein involve administering to a patient an effective amount of one or more CXCR7 compounds provided herein. In a preferred embodiment, the compound(s) of the invention are preferably administered to a patient (eg, a human) orally. Treatment regimens may vary depending on the compound used and the specific condition being treated; for the treatment of most disorders, a dosing frequency of 4 times daily or less is preferred. In general, a twice daily dosing regimen appears to be more preferred, with once daily dosing particularly preferred. However, it should be understood that the specific dose level and treatment regimen for any particular patient will depend on numerous factors, including the potency of the particular compound used, age, body weight, general health, sex, diet, time of administration, route of administration, rate of elimination, combination drugs (i.e. other drugs administered to the patient) and the severity of the specific disease being treated, as well as the opinion of the prescribing practitioner. In general, the use of the minimum dose sufficient to provide effective therapy appears to be preferable. Patients can usually be monitored for therapeutic effectiveness using medical or veterinary criteria appropriate for the condition being treated or prevented.

Depending on the cancer being treated and the condition of the subject, the compounds and compositions of the present invention can be administered via oral, parenteral (eg, intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection or implant), inhalation , nasal, vaginal, rectal, sublingual or local routes of administration, and they can be formulated, separately or together, into suitable dosage forms containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and carriers suitable for each route of administration. The present invention also contemplates administering the compounds and compositions of the present invention in a sustained absorption composition.

Dosage levels on the order of about 0.1 mg to about 140 mg per kilogram of body weight per day (about 0.5 mg to about 7 g per human patient per day) are useful. The amount of active ingredient that can be combined with carrier materials to form a unit dosage form will vary depending on the patient being treated and the particular route of administration. Unit dosage forms typically contain from about 1 mg to about 500 mg of active ingredient. Sufficient amounts of compounds should be administered to achieve serum concentrations of 50 to 200 ng/mL.

The compounds and compositions of the present invention can be combined with other compounds and compositions having related uses for the prevention and treatment of cancer. Such other drugs can be administered in a manner and in an amount usually used for them, simultaneously or sequentially with a compound or composition of the present invention. When a CXCR7 inhibitor is used concomitantly with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to the CXCR7 inhibitor is preferred. Accordingly, the pharmaceutical compositions of the present invention also include those that also contain one or more other active ingredients or therapeutic agents in addition to the CXCR7 inhibitor.

The additional therapeutic agent used in combination therapy, whether compound or antibody, can be administered via oral, parenteral (eg, intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection or implant), inhalation, nasal, vaginal, rectal, sublingual or local route of administration. In addition, the compounds and/or antibodies can be formulated individually or together in suitable unit dosage forms containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and carriers suitable for each route of administration. The present disclosure also contemplates administering the compounds and antibodies of the present disclosure in a sustained absorption composition.

It should be understood that the specific dose level and dosing frequency for any given patient may vary and will depend on numerous factors, including the activity of the particular compound(s) and/or antibodies used, the metabolic stability and duration of action of that compound, age, body weight, hereditary characteristics, general health, sex, diet, route and time of administration, rate of elimination, combination of drugs, severity of the particular condition and host being treated.

Combination therapy includes coadministration of a CXCR7 inhibitor and one or more additional therapeutic agents, sequential administration of a CXCR7 inhibitor and one or more additional therapeutic agents, or simultaneous administration of separate compositions such that one composition contains a CXCR7 inhibitor and one or more compositions containing one or more additional therapeutic agents. therapeutic agents.

Co-administration includes administration of a CXCR7 inhibitor of the present disclosure within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours after one or more administrations of one or more additional therapeutic agents. Moreover, the CXCR7 inhibitor and one or more additional therapeutic agents can be administered once daily or twice, three or more times daily to provide the preferred dosage level per day.

IV. Examples

The following examples are offered by way of illustration and not limitation of the claimed invention.

Example 1: FRS2β expressed in luminal progenitor cells creates a microenvironment favorable to breast tumorigenesis.

To study the role of FRS2β in vivo, we mutated the Frs2β gene in mice by targeting the gene. The mutant mice grew normally and were fertile without major abnormalities. Frs2β promoter activity was detected by β-galactosidase staining of mature female breast tissue that was heterozygous for the Frs2β mutant allele (Fig. 1A). Frs2β transcript abundance increased significantly during pregnancy and lactation, then decreased after weaning (3 weeks postnatal) during a regression period (data not shown). Using immunohistochemistry, we confirmed that FRS2β is expressed in several cells of the mammary lobules ( Fig. 1C ). The majority of FRS2β-positive cells were negative for phospho-histone H3, a nuclear marker of dividing cells, indicating that they proliferate more slowly than others, consistent with a negative role of FRS2β in cell proliferation ( Fig. 1C ). FRS2β was expressed in several cells that were positive for cytokeratin 18 (a marker of luminal cells) but not cytokeratin 14 (a marker of myoepithelial cells) (Fig. 1D). These data indicate that a small number of luminal cells in the mammary gland express FRS2β. On the other hand, complete staining of the mammary gland showed no major structural abnormalities in the mutant mice. This led us to study the pathological role of FRS2β in tumorigenesis.

We crossed Frs2β mutant mice with MMTV-neu(+) mice to generate MMTV-neu(+)/Frs2β(+/+) mice and MMTV-neu(+)/Frs2β(-/-) mice, hereafter referred to as mice Frs2β (+/+) and Frs2β (-/-), respectively. We observed that tumorigenesis began earlier with a greater likelihood in MMTV- neu (+) mice that underwent pregnancy at ~8 weeks of age (23.4 + 1.9 weeks, 83%, n = 8) than in parental MMTV- neu (+) mice (32.6 + 2.6 weeks, 23.4%, n = 8). Therefore, we examined tumorigenesis in mice immediately after pregnancy and lactation. We used nuclear magnetic resonance (NMR) imaging, which is a sensitive way to detect tumors even with a diameter of 1 mm20 (Fig. 1E). We began to observe the emergence of small tumors 5-8 weeks after the start of the measurement and found that the rate of tumor growth was much lower in Frs2β (-/-) mice than in Frs2β (+/+) mice (Figures 1E, 1F). while cancer incidence showed similar rates: 83.2% (n = 18) for Frs2β (+/+) and 88.2% (n = 17) for Frs2β (-/-). This suggests that FRS2β plays an important role in mammary tumorigenesis. To explore the molecular mechanisms, we first compared tumor histology. Frs2β(+/+) tumors had sufficient stroma reminiscent of human breast cancer tissue ( Fig. 1G ). However, it was much less present in Frs2β (-/-) tumors. Considering that tumor stroma is a major component of the tumor microenvironment, we hypothesized that FRS2β may play a role in creating a favorable microenvironment for tumorigenesis in breast tissues.

Histological examination revealed that Frs2β(+/+) tumors contained abundant stroma reminiscent of human breast cancer tissue (arrows in Fig. 1G). In contrast, very little stroma was observed in Frs2β(−/−) tumors. High levels of smooth muscle actin (SMA)-positive CAFs were present in the stroma of Frs2β(+/+) tumors but not in Frs2β(−/−) tumors (arrows in Fig. 1H). These results indicate that FRS2β is required for tumor stroma formation.

Testing the idea that FRS2β plays a role in establishing the breast tissue microenvironment required for tumorigenesis, even before tumor initiation, we performed xenograft experiments in which Frs2β(+/+) tumor cells were inoculated into young virgin precancerous breast tissue Frs2β (+/+) and Frs2β (-/-) mice. We cultured Frs2β(+/+) tumor cells in a serum-free suspension as beads to enrich CSC _s15,21 . We then inoculated them into 8-week-old virgin Frs2β (+/+) or Frs2β (−/−) mammary tissues after limiting dilution and measured tumorigenesis ( Fig. 2A ). Interestingly, tumors formed only in Frs2β (+/+) but not in Frs2β (−/−) mammary tissues and grew rapidly within 1 month (Figures 2B, 2C, 2D). This result suggests that CSCs disappeared in the microenvironment of Frs2β(−/−) mammary tissues. As expected, tumors did not form when Frs2β(+/+) tumor cells were inoculated into Frs2β(+/+) male breast fat pads (data not shown), suggesting that mammary tissues are important for tumorigenesis. Therefore, precancerous breast cells expressing FRS2β appear to create a microenvironment that supports CSC growth and promotes tumorigenesis.

By immunohistochemistry, we found similar numbers of luminal cells expressing FRS2β in MMTV-neu(−) mice and MMTV-neu(+) mice (Fig. 2E). Endogenous expression of ErbB2 was moderately reduced in FRS2β-positive cells from MMTV-neu(-) mice (yellow arrows), consistent with the fact that FRS2β is involved in the ubiquitylation and degradation of ErbB219; whereas ErbB2 was overexpressed in FRS2β-positive cells in MMTV-neu(+) mice (white arrows). To further examine which luminal cell type expresses FRS2β, we sorted mammary cells using surface markers. It is known that luminal cells are enriched in the CD49f _low /CD24 _high cell population and that luminal progenitor cells can be enriched by further CD61 fractionation to obtain the CD49f _low /CD24 _high /CD61 ₊ population. Significant FRS2β expression was observed in 23.6% of cells from the CD49f _low /CD24 _+high /CD61+ luminal progenitor cell population (Figure 2F). We confirmed that FRS2β is lost in a population of CD49f _low /CD24 _high /CD61+ progenitor cells derived from Frs2β (−/−) mammary cells. These data suggest that a subpopulation of luminal progenitor cells in the mammary gland expresses FRS2β.

Example 2: Precancerous Breast Cells Express Cytokines that are Dependent on FRS2β Expression

We next examined the molecular mechanisms by which FRS2β expressed in luminal progenitor cells creates a microenvironment favorable to tumorigenesis. We cultured Frs2β(+/+) or Frs2β(−/−) precancerous mammary cells in a serum-free suspension to enrich for undifferentiated or beaded progenitor cells and measured their ability to form the mammosphere (Figures 3A and 3B). We dissociated these primary spheres into a single cell suspension and cultured them to create secondary mammospheres. Secondary spheres are thought to accurately reflect the number of sphere-forming, undifferentiated, or progenitor cells. We found that FRS2β deficiency resulted in a significantly lower sphere-forming ability ( Figures 3A, 3B ). There was no significant difference in mammary gland diameter, suggesting that proliferation rates were similar between Frs2β(+/+) and Frs2β(−/−) precancerous mammary cells. To examine which luminal progenitor cell functions are impaired by loss of FRS2β, we compared transcriptomic profiles between Frs2β(+/+) and Frs2β(−/−) mammosphere cells using DNA microarray analysis. Gene set enrichment analysis (GSEA) revealed that a set of genes associated with stem cell function and a set of genes associated with interferon signaling were enriched in Frs2β(+/+) mammosphere cells compared to Frs2β(−/−) cells (Fig. .3C). GSEA in precancerous mammary epithelial cells also revealed that gene sets associated with NFκB targets, stem cell function, and stroma were enriched in Frs2β(+/+) cells compared to Frs2β(−/−) cells (Figure 3D). A set of genes associated with the ERK pathway are upregulated in Frs2β(−/−) cells compared with Frs2β(+/+) cells, which was expected because FRS2β inhibits ERK signaling. Many genes encoding cytokines were upregulated in Frs2β(+/+) cells; among them, 18 genes were expressed at more than 1.5-fold higher levels in Frs2β(+/+) cells than in Frs2β(−/−) cells (data not shown).

We then focused on IGF1, which is included in the set of genes associated with stem cell function, and on CXCL12, which is included in the set of genes associated with interferon signaling and in the set of genes associated with stroma, among the major genes that were highly expressed in Frs2β (+/+) cells. Quantitative PCT analysis (qPCT) confirmed that Igf1 and Cxcl12 transcripts were more highly expressed in heterozygous Frs2β(+/−) mammary cells than in Frs2β(−/−) cells, whereas differentiated cell markers (Keratin8, Keratin18, and Keratin14) were upregulated in Frs2β(−/−) cells (Fig. 3E). Using immunohistochemistry, we confirmed that the protein levels of IGF1, CXCL12, and αSMA, a CAF marker, were higher in Frs2β(+/+) mammary tissues (Figure 3F). Strong αSMA staining confirmed CAF mobilization in wild-type mammary tissues.

Example 3: FRS2β-Dependent Increase in CXCL12 Production in Precancerous Breast Cells Allows Tumor Treatment with a CXCR7 Inhibitor or a CXCR7 Inhibitor in Combination with Another Tumor Growth Modulating Therapeutic

The formation of a tumor sphere reflects the properties of CSCs, the growth of which depends on cytokines in culture. To determine whether IGF1 derived from precancerous mammary epithelial cells plays a role in tumor sphere formation, we cultured Frs2β(+/+) tumor cells under serum-free suspension conditions without a cytokine cocktail in the presence or absence of precancerous mammary epithelial cells Frs2β( +/+) (Fig. 4A). We observed the formation of tumor spheres by Frs2β(+/+) tumor cells in the presence of, but not in their absence, precancerous Frs2β(+/+) mammary epithelial cells (compare control IgG with untreated [NT] in Figures 4B and 4C). Treatment with IGF1 neutralizing antibody (IGF1 NAb) significantly reduced tumor sphere formation by Frs2β(+/+) tumor cells cocultured with Frs2β(+/+) precancerous breast cells (Figures 4B and 4C). These observations indicate that IGF1, derived from adjacent Frs2β(+/+) premalignant mammary epithelial cells, plays an important role in the formation of tumor spheres. Thus, IGF1 derived from premalignant Frs2β(+/+) mammary epithelial cells may support CSC growth.

To test whether precancerous mammary epithelial cell-derived CXCL12 plays a role in cancer-associated fibroblasts (CAFs), we cocultured Frs2β(+/+) CAFs with Frs2β(+/+) or Frs2β(-/-) precancerous mammary epithelial cells (Fig. 4D). We confirmed that Cxcl12 expression levels were higher under these culture conditions in Frs2β(+/+) precancerous mammary cells than in Frs2β(−/−) cells (Figure 4E). We observed significantly more migrated CAFs when cocultured with Frs2β(+/+) precancerous breast cells than with Frs2β(−/−) cells (Figures 4F and 4G). CXCL12 binds to CXC receptor (CXCR) 4 and CXCR7. We did not observe a significant effect on CAF mobilization when treated with CXCR4 inhibitor at the reported optimal concentration: AMD3100 (100 μg/ml) or compound 1 (100 μg/ml) alone (data not shown); whereas, when treated with a combination of both inhibitors, CAF mobilization was significantly reduced in a dose-dependent manner (Figures 4H, 4I). These observations suggest that CXCL12, derived from adjacent Frs2β(+/+) precancerous breast cells, plays an important role in CAF mobilization. Therefore, it appears that FRS2β-dependent increased production of cytokines, including IGF1 and CXCL12, in precancerous breast cells allows for CSC maintenance and CAF mobilization.

What are the molecular mechanisms that induce IGF1 and CXCL2 expression in precancerous breast tissue? Because Igf1 and Cxcl12 were included in the set of NFkB target genes ( Fig. 3D ), and the AKT–NFkB axis is activated by many signaling pathways that include ErbB2 and CXCL12, we examined whether NFkB activation is involved in the production of these cytokines. To this end, we cultured precancerous Frs2β(+/+) mammary epithelial cells and treated them with DHMEQ, a specific NFkB inhibitor ( Fig. 5A ). DHMEQ treatment inhibited the expression of Igf1, Cxcl12, and IkBα, a well-known NFkB-inducible gene, in a dose-dependent manner (Fig. 5B), suggesting that NFkB activation plays an important role in the expression of IGF1 and CXCL12 in precancerous mammary epithelial cells.

We next examined the activation of the AKT-NFkB axis in premalignant breast tissues in vivo. Immunoblotting of lysates from precancerous breast tissues revealed higher levels of phosphorylated AKT, higher amounts of the NFkB components RelA and RelB in the nucleus, higher levels of phosphorylated IKKb, and lower levels of IkBa in Frs2β (+/+) tissues relative to Frs2β (-/ -) (Figs. 5C–5E). As expected, phosphorylated ERK1/2 was present at reduced levels in Frs2β(+/+) tissues than in Frs2β(−/−) tissues (Fig. 5C). Moreover, immunohistochemistry showed that RelA is localized to the nucleus in a much higher proportion of Frs2β(+/+) than Frs2β(−/−) in premalignant luminal cells (Fig. 5G, red arrows in left panel and middle panel). DHMEQ treatment in vivo dramatically reduced the number of precancerous luminal Frs2β(+/+) cells bearing RelA in the nucleus (Fig. 5F and G, right panel) and inhibited the expression of Igf1 and Cxcl12 transcripts in precancerous breast tissues (Fig. 5H). These results suggest that NFκB activation in precancerous luminal cells plays an important role in the expression of IGF1 and CXCL12 in precancerous mammary epithelial cells in vivo. It appears that FRS2β triggers the AKT-NFkB axis in premalignant luminal cells, thereby inducing the production of cytokines including IGF1 and CXCL12, which in turn activate NFkB in an autocrine or paracrine manner to spread the effects of NFkB activation to the surrounding mammary epithelial cell layer .

To test whether IGF1 and CXCL12, expressed in the precancerous mammary microenvironment, contribute to tumorigenesis, we treated Frs2β (+/+) mice with IGF1 neutralizing antibodies and/or a combination of a CXCR4 inhibitor and a CXCR7 inhibitor (compound 1) (both together, inhibitor CXCL12) after inoculation of Frs2β (+/+) tumor cells. Treatment with either an IGF1 neutralizing antibody or a CXC12 inhibitor significantly reduced tumorigenesis, and combination treatment with both an IGF1 neutralizing antibody and CXCL12 inhibitors showed the greatest inhibitory effect on tumor volumes and weights (Figures 5I-5K). Body weight did not change significantly (data not shown), indicating no toxic effects. These results indicate that FRS2β-dependent increased production of IGF1 and CXCL12 in premalignant breast tissues creates a microenvironment that is important for tumorigenesis.

We next examined FRS2β expression in breast tumors. Immunohistochemistry showed that cells expressing FRS2β were present in the breast tumors (Fig. 6A). The expression levels of Igf1 and Cxcl12 were higher in Frs2β(+/+) tumors than in Frs2β(−/−) tumors (Fig. 6B). Immunohistochemistry confirmed that the expression levels of IGF1 and CXCL12 were higher in Frs2β(+/+) tumors than in Frs2β(−/−) tumors (Fig. 6C). Therefore, it is reasonable to speculate that FRS2β triggers the AKT-NFkB axis to induce the production of IGF1 and CXCL12 in tumor tissues.

Finally, we examined FRS2β expression in human breast cancer tissues using immunohistochemistry. FRS2β expression levels varied among cancer cells (Fig. 6D). Breast cancer tissues in which FRS2β expression levels were high (+++) had significantly higher levels of cancer stroma than tissues with intermediate (++) or low (+) FRS2β expression levels (p = 0.0499, test Barnard) (Fig. 6E). Additionally, analysis of published gene expression profiles revealed that patients with higher levels of FRS2β expression in breast cancer tissues had a worse prognosis ( Figure 6F ).

In this study, we demonstrated that FRS2β protein is expressed in a subset of luminal cells and triggers the production of cytokines, including IGF1 and CXCL12. FRS2β can stimulate the AKT–NFkB axis, promoting cytokine production while suppressing ERK signaling. It appears that these cytokines, in turn, activate NFκB in the surrounding luminal cells of the mammary gland in an autocrine or paracrine manner, resulting in the creation of a cytokine-rich precancerous microenvironment that includes some stroma before tumor emergence ( Fig. 6G , top left panel). Once CSCs emerge in the precancerous microenvironment, they may be able to self-renew in the presence of IGF1 and produce tumor cells via CXCL12-mobilized stromal cells, which subsequently become CAFs. CSCs and tumor cells can produce IGF1 and CXCL12 on their own, leading to rapid growth and tumorigenesis ( Fig. 6G , lower left panel). Without FRS2β, cytokines remain at low levels and the corresponding precancerous microenvironment is not created (Fig. 6G, top right panel); even when CSCs emerge, they fail to grow efficiently ( Fig. 6G , lower right panel). Based on these results, we suggest that FRS2β represents a promising target for breast cancer prevention. In addition, we showed that combination therapy targeting IGF1 and CXCL12 effectively prevents early-stage tumorigenesis.

The tumor microenvironment is composed of various cell types: CAFs, mesenchymal stem cells, bone marrow dendritic cells, immune cells, and new blood vessels (3). On the other hand, it remains unclear which cell types in the precancerous microenvironment contribute to tumorigenesis. Herein, we found that luminal cells and luminal progenitor cells are an important cell type in the precancerous microenvironment, and that FRS2β, expressed in luminal cells and luminal progenitor cells, plays a critical role in the production of cytokines, resulting in the creation of a cytokine-rich precancerous microenvironment, which is important for tumor development.

Although the foregoing invention has been described in some detail by way of illustrations and examples for the purpose of clarity of understanding, one skilled in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each link were individually incorporated by reference. If there is a conflict between this application and a reference herein, the application currently pending will control.

Claims (34)

1. A method of treating breast cancer in an individual in need thereof, wherein the method comprises administering to the individual a CXCR7 inhibitor, wherein the individual has aberrant expression of FRS2β, and wherein The CXCR7 inhibitor has a Formula I or Formula II structure: or pharmaceutically acceptable salts thereof, wherein R ² and R ³ each represent H; C ¹ represents quinolinyl, which is optionally substituted with 1 to 3 R ⁴ substituents; C ² is selected from the group consisting of thiazole, pyrazole and oxazole, each of which is optionally substituted with 1 to 2 R ⁵ substituents; C ³ is selected from the group consisting of cyclohexyl, piperidinyl and phenyl, each of which is optionally substituted with 1 to 2 R ⁶ substituents; each R ⁴ is independently selected from the group consisting of methyl, ethyl, isopropyl, 2-fluoroethyl, 2-fluoroisopropyl, 2-hydroxyisopropyl, methoxy, chloro, CO ₂ H, -CH ₂ CO ₂ H, XCO ₂ H; each R ⁵ is independently selected from the group consisting of methyl, fluoro, chloro, CO ₂ H, and CH ₂ CO ₂ H; each R ⁶ is independently selected from the group consisting of methyl, fluoro, chloro, -OH, CO ₂ H, and CH ₂ CO ₂ H; And each X represents a linker group having a formula selected from the group consisting of -OCH ₂ -, -OCH ₂ CH ₂ -, -OCH ₂ CH ₂ CH ₂ -; or or pharmaceutically acceptable salts thereof, wherein bicyclic part having X^a, X^band X^cas vertices of the ring, selected from: R ² is selected from the group consisting of H and C _1-8 alkyl; R ³ represents H; Z represents each Q represents N; R ⁵ is aryl, optionally further substituted with 1-3 R ^a substituents; And each R ^a is halogen or C _1-8 alkyl. 2. The method according to claim 1, wherein the CXCR7 inhibitor has a structure selected from the group consisting of or a pharmaceutically acceptable salt thereof. 3. The method of claim 1, wherein the individual expresses FRS2β in one or more luminal progenitor cells. 4. The method of claim 1, wherein the individual expresses FRS2β in one or more luminal progenitor cells of the mammary gland. 5. The method according to claim 1, further comprising administering an additional therapeutic agent. 6. The method of claim 1, further comprising administering therapeutically effective amounts of an anti-IGF1 antibody and a CXCR4 inhibitor. 7. The method of claim 1, further comprising administering a therapeutically effective amount of an anti-IGF1 antibody. 8. The method of claim 1, further comprising administering a therapeutically effective amount of a CXCR4 inhibitor. 9. The method of claim 1, wherein the subject is diagnosed with aberrant expression of FRS2β before administration of the CXCR7 inhibitor or additional therapeutic agent.