CXCR4 Protein Epitope Mimetic Antagonist POL5551 Disrupts Metastasis and Enhances Chemotherapy Effect in Triple-Negative Breast Cancer

Abstract

The SDF-1 receptor CXCR4 has been associated with early metastasis and poorer prognosis in breast cancers, especially the most aggressive triple-negative subtype. In line with previous reports, we found that tumoral CXCR4 expression in patients with locally advanced breast cancer was associated with increased metastases and rapid tumor progression. Moreover, high CXCR4 expression identified a group of bone marrow-disseminated tumor cells (DTC)-negative patients at high risk for metastasis and death. The protein epitope mimetic (PEM) POL5551, a novel CXCR4 antagonist, inhibited binding of SDF-1 to CXCR4, had no direct effects on tumor cell viability, but reduced migration of breast cancer cells in vitro. In two orthotopic models of triple-negative breast cancer, POL5551 had little inhibitory effect on primary tumor growth, but significantly reduced distant metastasis. When combined with eribulin, a chemotherapeutic microtubule inhibitor, POL5551 additively reduced metastasis and prolonged survival in mice after resection of the primary tumor compared with single-agent eribulin. Hypothesizing that POL5551 may mobilize tumor cells from their microenvironment and sensitize them to chemotherapy, we used a "chemotherapy framing" dosing strategy. When administered shortly before and after eribulin treatment, three doses of POL5551 with eribulin reduced bone and liver tumor burden more effectively than chemotherapy alone. These data suggest that sequenced administration of CXCR4 antagonists with cytotoxic chemotherapy synergize to reduce distant metastases.

Figure 1. High CXCR4 expression associated with high rate of metastases even in patients without detectable bone marrow disseminated tumor cells

A) Overall survival of patients with primary breast tumors that are CXCR4^High (n=10) and CXCR4^Low (n=28); B) Overall survival of patients that are DTC^negative characterized by CXCR4^High (n=5) or CXCR4^Low (n=15) expression of the primary tumor

Figure 2. CXCR4 selective antagonist POL5551 decreases breast cancer cell migration and promotes anoikis in vitro

A) Complementation imaging. Binding of SDF-1-CGLuc to NGLuc-CXCR4 or NGLucCXCR7 reconstitutes GLuc, producing light as a quantitative measure of ligand:receptor binding. SDF-1-CGLuc MDA-MB-231 cells were co-incubated overnight with NGLuc-CXCR4 or NGLuc-CXCR7 MDA-231 cells, followed by incubation with various concentration of POL5551 for 6 hours (***, p<0.001); B) MDA-231 cells were incubated with various concentration of POL5551 for 48 hours and cell viability was assessed by an MTT assay; C) & D) A wound gap was created by scratch in confluent monolayers of SDF-1-secreting MDA-231 cells. Gap closure was measured after 24 hours of treatment with various concentrations of POL5551 (*, p<0.05; ***, p<0.001); E) & F) Cell survival after forced suspension culture with SDF-1 (12.5ng/ml; 50ng/ml) and POL5551 (8uM). Cell colonies were stained with crystal violet. The absorbance of crystal violet dye dissolved with 1% SDS was measured by plate reader at 570–630nm (*, p<0.05; **, p<0.01; ***, p<0.001).

Figure 3. CXCR4 antagonist POL5551 decreases metastasis but has little effect on primary MFP tumor growth in a xenograft NSG model

A) MDA-MB-231 mammary fat pad orthotopic model schema: MDA-MB-231 tumor cells were injected to the mammary fat pad of mice. Treatment with POL5551 (20mg/kg, s.c., b.i.d, n=3) or saline (n=5) commenced on day 8 when the tumor became palpable and continued until the end of study; B) & C) Primary MFP tumor growth as measured by bioluminescence imaging, representative image showing the primary tumors on Day 21 ; D) & E) Distant metastasis to the chest as measured by bioluminescence imaging, representative image showing the distant metastases on Day 41 (*, p<0.05); F) Human cytokeratin 18 (CK18) staining of MDA-MB-231 tumor cells in lung tissue; G) Quantification of CK18 positive cells in lung tissue; ***, p<0.001; H) Ex vivo BLI of bone metastases (*, p<0.05); I) Ex vivo BLI of liver metastases (p>0.05).

Figure 4. POL5551 monotherapy decreased distant metastasis in a disseminated tumor model

A) intracardiac tumor injection model, 4T1-Luc murine tumor cells were injected into the left ventricle of the BALB/c mice. Treatment with POL5551 (20mg/kg, s.c., b.i.d, n=7) or vehicle (saline, n=6) was from day 3 to day 11. Mice were sacrificed on day 11; B) & C) Bioluminescent imaging of 4T1-Luc tumor burden in leg bones (ns, p=0.23) and whole body (ns, p=0.08); D) H&E staining of kidney tissue, 4T1 tumor foci/nodules are outlined in white to increase visibility; E) Quantification of 4T1 tumor foci in kidney per section (n=6/group; **, p<0.01); F) & G) μCT analysis for calculation of trabecular bone to tissue volume ratio (BV/TV) (n=6/group; *, p<0.05).

Figure 5. Combination of eribulin with POL5551 in the neoadjuvant setting improves survival compared to eribulin alone

A) Experimental design: MDA-MB-231 cells were mixed with matrigel and implanted s.c. into the right flank of the NSG mice. Ten days after tumor implantation, mice (n=10/group) were treated as follow: a) Vehicle saline (i.v.) on day 10, 17 & 24) plus (s.c., b.i.d) from day 10 to the day of surgery; b) Eribulin alone (0.1mg/kg, i.v) on days 10, 17 and 24; c) Eribulin treatment (days 10, 17 & 24) plus POL5551 (20mg/kg, s.c., b.i.d) administration daily from day 10 until termination; B) Primary tumor size measured by caliper; C) Primary tumor weight at surgery (ns, p>0.05); D) Bioluminescence imaging of distant metastasis to the chest (*, p<0.05); E) Bioluminescence imaging of distant metastasis to the bone in eribulin alone and combo group (***, p<0.001); F) Kaplan Meier survival curve.

Figure 6. Reduction of bone tumor burden: POL5551 in combination with eribulin is superior to eribulin alone

A) Schema of bone metastasis model utilizing “framing dose” strategy: MDA-MB-231 bone metastases were established by intracardiac inoculation in NSG mice, and on day 10 mice were treated as following: a) Vehicle (n=4): saline (s.c. and i.v.); b) Eribulin alone (n=5): eribulin (0.2mg/kg, i.v.) alone and saline (s.c); c) Combination treatment (n=5): POL5551 (20mg/kg, s.c.) was administered 4 hours before eribulin and 4- and 18- hours after eribulin (0.2mg/kg, i.v.) chemotherapy; B) & C) Bioluminescent imaging of tumor burden in liver and bone (*, p<0.05; ***, p<0.001); D) Representative X-Ray images of tumor associated osteolytic lesion in leg bone; E) Quantification of osteolytic lesion area; F) Representative image showing extent of metastatic burden in liver and leg bone on day17 (*, p<0.05; **, p<0.01); G) Mouse weight lost between day 9 and day 17. Statistical analyses are as compared to vehicle treatment (ns, p>0.05; **, p<0.01).