JLF and PB provided leukemic samples

JLF and PB provided leukemic samples. xenograft (PDX) models, we found that NG2 is crucial for MLLr-B-ALL engraftment upon intravenous (i.v.) transplantation. In vivo blockade of NG2 using either chondroitinase-ABC or an anti-NG2-specific monoclonal antibody (MoAb) resulted in a significant mobilization of MLLr-B-ALL blasts from bone marrow (BM) to peripheral blood (PB) as demonstrated by cytometric and 3D confocal imaging analysis. When combined with either NG2 antagonist, VxL treatment achieved higher rates of complete remission, and consequently higher EFS and delayed time to relapse. Mechanistically, anti-NG2 MoAb induces neither antibody-dependent cell-mediated not complement-dependent cytotoxicity. NG2 blockade rather overrides BM stroma-mediated chemoprotection through PB mobilization of MLLr-B-ALL blasts, thus becoming more accessible to chemotherapy. We provide a proof of concept for NG2 as a therapeutic target for MLLr-B-ALL. test, as appropriate. For MRD studies, data are expressed as median (range) and significant differences were analyzed by the median test. CRRs were PROTAC ERRα ligand 2 statistically compared using Pearsons value? ?0.05. Results NG2 blockage does not elicit cytotoxicity of MLLr-B-ALL blasts but results in in vivo robust blast mobilization into PB We first interrogated the ability of FACS-sorted NG2+ and NG2? diagnostic leukemic blasts to reproduce the B-ALL phenotype by engrafting in NSG mice. When equal numbers of NG2+ and NG2? B-ALL primary blasts (105 cells/patient, em n /em ?=?4) were intravenously (i.v.) infused into NSG mice, engraftment levels after 8 weeks were 3.5-fold higher in the PB of mice transplanted with NG2+ PROTAC ERRα ligand 2 than in equivalent mice transplanted with NG2? blasts (11.2??1.8% vs. 3.2??1.5%, em p /em ?=?0.008) (Fig.?1a, left and middle panels). Overnight in vitro treatment of NG2+ B-ALL blasts with either Chase (0.1U/ml) or anti-NG2 MoAb (either 7.1 (0.7?g/ml) or 9.2.27 (100?g/ml) clone) abolished their engraftment after i.v. transplantation (12??1.7% vs. 2%, em p /em ? ?0.001) (Fig.?1a, right panel). Importantly, clinical data from MLLr-BCP-ALL infants ( em n /em ?=?55) uniformly enrolled in the Interfant treatment protocol reveals that patients with refractory or relapsed disease initially had at diagnosis 28% more NG2-expressing blasts than those patients not experiencing relapse (53% vs. 41%, em p /em ? ?0.05). This suggests that NG2 plays a role in vivo in the propagation of MLLr-B-ALL cells. Open in a separate window Fig. 1 In vivo blockade of NG2 results in the robust mobilization of MLLr-B-ALL blasts into PB. a Left, experimental design of the in vitro treatment with NG2 antagonists. Middle, engraftment capacity of PROTAC ERRα ligand 2 NG2+ or NG2? MLLr-B-ALL blasts 8 weeks after i.v. transplantation. Right, overnight in vitro exposure of NG2+ blasts to the NG2 antagonists Chase, 7.1 MoAb, or 9.2.27 MoAb abolishes their engraftment potential [18]. b Experimental design of the in vivo treatment with NG2 antagonists. c Monitoring of the levels of leukemic grafts in PB and BM before (day 0) and after (day 7) the indicated treatments. Each line represents the same mouse before and after treatment. d Levels of leukemic grafts in BM (top panel) and PB (bottom panel) after the indicated in vivo treatments. Results are shown as mean??SEM, relative to day 0 (before treatment). e Representative FACS plots showing the identical leukemia NG2+ phenotype in both diagnostic samples and primografts. The right panel shows the in vivo effectiveness of the 7.1 MoAb, PROTAC ERRα ligand 2 which abolishes NG2 expression in blasts recovered from primografts. em n /em , 8C12 mice/group from three different patients. * em p /em ? ?0.05; ** em p /em ? ?0.01; **** em p /em ? ?0.0001 We then assessed in vitro and in vivo whether anti-NG2 (7.1 MoAb) effectively eliminates NG2+ MLLr-B-ALL blasts. In vitro, anti-NG2 treatment was unable Rabbit Polyclonal to MAD2L1BP to activate effector mechanisms including CDC (Fig.?2a, b) and ADCC (Fig.?2c), resulting in no cytotoxicity of NG2-expressing MLLr-B-ALL cells in 4- and 24-h assays. As a PROTAC ERRα ligand 2 control, Rituximab (anti-CD20 MoAb) effectively activated both CDC and ADCC cytotoxicity mechanisms to eliminate Daudi cells (Fig.?2). We further addressed whether NG2 blockage elicits cytotoxicity in vivo, thus impacting the engraftment dynamics of MLLr-B-ALL blasts in PDX models (Fig.?1b). A total of 105 NG2+?MLLr-B-ALL cells ( em n /em ?=?4 patients) were i.v. transplanted and, once PB engraftment reached 0.5%, mice were daily i.p. treated with the NG2 antagonist Chase (0.06?U/mouse) or anti-NG2 7.1 MoAb (10?mg/kg/mouse) for 7 days (Fig.?1b). In line with the in vitro data, NG2 blockage in vivo failed to reduce tumor burden (Fig.?1c). However, when we compared vehicle-treated primografts with primografts treated with either NG2 antagonist after completion of the treatment, we found that treatment with NG2 antagonists significantly reduced leukemic burden in BM that was accompanied by a massive leukemia infiltration in PB ( em p /em ? ?0.01; Fig.?1c, d). Importantly, PDX models reproduced the immunophenotype of the de novo primary leukemia, and blasts recovered from primografts treated with NG2 blockers were mainly NG2? (Fig.?1e). These results show that blocking NG2 in vivo mobilizes MLLr-B-ALL blasts to PB. This coupled to our previous data [18], reporting similar engraftment levels.