Using the MOLM-14/Luc/GFP xenograft model, mice were administered daily treatment with FLT3 and CXCR4 inhibitors after achieving high levels of leukemia chimerism in the blood (Determine S4A). LY2510924 revealed that genes related to TGF- signaling may confer resistance against the drug combination. In co-culture experiments of FLT3-ITD-AML and stromal cells, both silencing of TGF- in stromal cells or TGF–receptor kinase inhibitor enhanced apoptosis by combined treatment. Disruption of the CXCL12/CXCR4 axis in FLT3-ITD-AML by LY2510924 and its negligible effects on normal immunocytes could safely enhance the potency of quizartinib, which may be further improved by blockade of TGF- signaling. < 0.05, ** < 0.01. 2.2. CXCR4 Inhibition by LY2510924 Significantly Reverses Stroma-Mediated Resistance to Quizartinib In Vitro, Mainly Through the MAPK Pathway To determine the combined effects and mechanisms of the CXCR4 blockade by LY2510294 with FLT3 inhibitors, we next tested whether CXCR4 inhibition by LY2510924 could overcome stroma-mediated protection against quizartinib in FLT3-ITD mutated AML cells in vitro by co-culturing MOLM-14 cells with MS-5 stromal cells or hMSC from FLT3-ITD-AML (Table S1) for three days. CXCR4 binding to 12G5 antibody was blocked by LY2510924 (Physique 2A,C) in both culture systems, with or without stromal cells. The quizartinib-induced apoptosis of AML cells was significantly reduced by stromal cells, and this protective effect of stromal cells was reduced by LY2510924 (Physique Desacetylnimbin 2B,D). Open in a separate window Physique 2 LY2510924 reverses stroma-mediated resistance to Desacetylnimbin quizartinib mainly through the MAPK pathway. (ACD) MOLM-14 cells were cultured alone (mono-culture) or co-cultured with stromal cells (MS-5 and hMSC from FLT3-ITD-AML) as indicated in the Materials and Methods. Mono-cultured and co-cultured cells were treated for 72 h with 1.0 nM quizartinib in the presence or absence of 1 M LY2510924. Surface CXCR4 12G5 staining (A,C) and percentages of apoptotic cells (B,D) were assessed by circulation cytometry. All results are expressed as the mean SD. * < 0.05, ** < 0.01. (E) After four hours of incubation with different doses of quizartinib in the presence or absence of 1 M LY2510924, MOLM-14 cells were harvested, and phosphorylation of FLT3, STAT5, AKT, ERK, and rpS6 (ribosomal protein S6) were detected by Western blot analysis. GAPDH was used as a loading control. Given that LY2510924 reduced stroma-mediated resistance to quizartinib, we next tested the effects of LY2510924/FLT3 inhibitor combination on Desacetylnimbin FLT3 signaling by studying the phosphorylation of FLT3 and downstream proteins in the FLT3 signaling pathway. FLT3 inhibition by quizartinib induced de-phosphorylation of FLT3 and downstream proteins in mono-culture system (Physique 2E). Co-culture with stromal cells experienced no significant effect on the phosphorylation of FLT3 by quizartinib. In terms of the downstream proteins, different effects of co-culture with stromal cells around the expression of AKT and ERK were seen in response to FLT3 inhibition. In the presence of stromal cells, FLT3 inhibition still induced AKT de-phosphorylation, but ERK phosphorylation was not fully inhibited, consistent with previous findings by Yang et al. [21]. However, CXCR4 inhibition by LY2501924 induced de-phosphorylation of Desacetylnimbin ERK, even in the presence of stromal cells, which was supported by inhibition of phosphorylation of the ribosomal protein S6 (rpS6). rpS6 is known to be directly phosphorylated through activation of p90 ribosomal S6 kinase by MAPK pathway in FLT3-ITD-AML [22] as well as activation of p70-S6 kinase 1 by PI3K/AKT/mTOR pathway. Thus, the anti-apoptotic effects of BM stroma appear to correlate with the prolonged activation of ERK, which could be effectively reversed by disruption of the CXCL12/CXCR4 axis by LY2510924. 2.3. LY2510924 Enhances Anti-Leukemia Effects in Combination with Quizartinib In Vivo To test the anti-leukemia efficacy of LY2510924 in combination with quizartinib in vivo, we injected MOLM-14 cells into non-irradiated NSG mice. Mice were randomized into four cohorts, which received the following treatment on day 5 post cell injection: Vehicle, quizartinib only, LY2510924 only, or the combination of LY2510924 and quizartinib for 21 times. Bioluminescence imaging (BLI) proven significantly decreased leukemic Gpr68 burden in every treated groups in comparison to settings (Shape 3A,B). Solitary agent therapy with quizartinib and LY2510924 decreased AML tumor burden, displaying comparable results on day time 19, quizartinib became far better than LY2510924 after that, and the mixture was most reliable. On day time 20, after fourteen days of daily treatment, three mice had been euthanized in each mixed group, and movement cytometry of circulating leukemic cells, in BM, and spleens exposed significant blockade of CXCR4 12G5 staining by LY2510924 in.