Inhibition of CXCR7 Extends Survival Following Irradiation of Brain Tumors in Mice and Rats

Supplemental Information

Inhibition of CXCR7 extends survival following irradiation of brain tumors in mice and rats.

Matthew J. Walters1,Karen Ebsworth1, Robert D. Berahovich1, Mark E.T. Penfold1, Shie-Chau Liu2,Reem Al Omran2, Mitomu Kioi2, Sophia B. Chernikova2, Diane Tseng2, Erin E. Mulkearns-Hubert3, Maksim Sinyuk3, Richard M. Ransohoff4,5, Justin D. Lathia3,5, Jason Karamchandani6, Holbrook E. K. Koort7, Penglie Zhang1, Jay P. Powers1, Juan C. Jaen1, Thomas J. Schall1, Milton Merchant8, Lawrence Recht8 and J. Martin Brown2

Supplemental Figure 1. CCX662 is a potent inhibitor of CXCR7 in vitro.A. CCX662 inhibits the binding of [125I]-labeled CXCL12 to 435-CXCR7 cells in buffer with an IC50 value of 9 nM.B.CCX662 inhibits the binding of [125I]-CXCL12 to 435-CXCR7 cells in 100% human serum with an IC50value of 18 nM. C.CCX662 inhibits the binding of [125I]-CXCL12 to NC-37 cells,which endogenously express CXCR7, with an IC50value of 7 nM. D. CCX662 does not inhibit CXCL12-induced calcium mobilization in CXCR4-expressing human activated lymphocytes.E.CCX662 induces the association of CXCR7 with-arrestin-2 with an EC50value of 3 nM. F.CCX662 inhibits the CXCL12-mediated migration of NC-37 cells through an endothelial cell monolayer in culture medium with an IC50value of 100 nM.G. CCX662 inhibits the binding of [125I]-CXCL12 to rat kidney cells in 100% rat serum with an IC50value of 14 nM.

Supplemental Figure 2. Inhibition of CXCR7 with CCX2066 significantly increases post-IR survival time in ENU-treated rats. A. Rats were exposed to the carcinogen ENU and, on day 115, given 20 Gy whole-brain IR. Immediately following IR, rats were dosed orally with 50 mg/kg CCX2066 or its vehicle (1% hydroxypropyl methylcellulose)once-daily for 3 weeks. CCX2066significantly increased median post-IR survival time (p<0.05 compared to vehicle, logrank test; n = 5-6 rats). Representative data from 2 separate experiments shown. B. Time-course of CCX2066 plasma levels after oral dosing of rats at 50 mg/kg. The dotted line depicts the rat plasma CCX2066 IC90 value, i.e. the concentration of CCX2066 required to inhibit 90% of CXCR7 molecules in rat plasma. Note that the 50 mg/kg dose results in plasma levels of CCX2066 in excess of the IC90 value for 24 hours.

Supplemental Figure 3. C6 GBM cells do not express CXCR7 protein. Expression of CXCR7 was assessed by [125I]-CXCL12 radioligand binding assay (A) and flow cytometry with CXCR7 mAb clone 11G8 (B). Control cell lines: “435” MDA MBs 435 cells; “435-X7” MDA MBs 435 cells transfected with a human CXCR7 expression vector.A. 435-X7 cells, but not C6 cells, bind to the [125I]-CXCL12 tracer. 435-CXCR7 cells exhibit the CXCR7 profile because [125I]-CXCL12 binding is inhibited by CXCL12/SDF, CXCL11/ITAC or CXCR7-specific inhibitor CCX771, but not by the inactive analog CCX704 or the CXCR4 antagonist AMD3100. B. 435-X7 cells, but not C6 cells, bind to the CXCR7 mAb (purple filled histogram). Green empty histogram shows binding of the isotype control mAb.