Bone marrow-derived mesenchymal stromal cells promote resistance to tyrosine kinase inhibitors in chronic myeloid leukemia via the IL-7/JAK1/STAT5 pathway

Abstract

Chronic myeloid leukemia (CML) is caused by the fusion of the BCR activator of RhoGEF and GTPase activating protein (BCR) and ABL proto-oncogene, the nonreceptor tyrosine kinase (ABL) genes. Although the tyrosine kinase inhibitors (TKIs) imatinib (IM) and nilotinib (NI) have remarkable efficacy in managing CML, the malignancies in some patients become TKI-resistant. Here, we isolated bone marrow (BM)-derived mesenchymal stem cells (MSCs) from several CML patients by Ficoll-Hypaque density-gradient centrifugation for coculture with K562 and BV173 cells with or without TKIs. We used real-time quantitative PCR to assess the level of interleukin 7 (IL-7) expression in the MSCs and employed immunoblotting to monitor protein expression in the BCR/ABL, phosphatidylinositol 3-kinase (PI3K)/AKT, and JAK/STAT signaling pathways. We also used a xenograft tumor model to examine the in vivo effect of different MSCs on CML cells. MSCs from patients with IM-resistant CML protected K562 and BV173 cells against IM- or NI-induced cell death, and this protection was due to increased IL-7 secretion from the MSCs. Moreover, IL-7 levels in the BM of patients with IM-resistant CML were significantly higher than in healthy donors or IM-sensitive CML patients. IL-7 elicited IM and NI resistance via BCR/ABL-independent activation of JAK1/STAT5 signaling, but not of JAK3/STAT5 or PI3K/AKT signaling. IL-7 or JAK1 gene knockdown abrogated IL-7-mediated STAT5 phosphorylation and IM resistance in vitro and in vivo Because high IL-7 levels in the BM mediate TKI resistance via BCR/ABL-independent activation of JAK1/STAT5 signaling, combining TKIs with IL-7/JAK1/STAT5 inhibition may have significant utility for managing CML.

Keywords: cancer; cell biology; cell death; chronic myelogenous leukemia (CML); cytokine; drug resistance; kinase cascade; mesenchymal stem cells (MSCs); myeloproliferative disease.

Figure 1.

Morphology and immunophenotypes of primary MSCs. A, primary NCMSCs on day 4, ×20. B, primary NCMSCs on day 8, ×20. C, primary NCMSCs on day 12, ×20. D, primary NCMSCs on day 20, ×20. E, confluent SMSCs, ×20. F, confluent RMSCs, ×20, scale bar, 50 μm. G, immunophenotypes of primary MSCs. H, cell proliferation kinetics of second passage primary NCMSCs, SMSCs, and RMSCs. I, fusion time of primary NCMSCs, SMSCs, and RMSCs. *, p < 0.05 compared with NCMSCs.

Figure 2.

RMSCs protect K562 and BV173 cells from TKI treatment. A, proliferation of K562/BV173 cells cocultured with different concentrations of MSCs after 24 h. B, proliferation of K562/BV173 cells cocultured with MSCs at different times. The ratio of MSCs and K562/BV173 cells was 1:1. C, proliferation of K562/BV173 cells cocultured with different MSCs after IM treatment. The ratio of MSCs and K562/BV173 cells was 1:1. After K562/BV173 cells were cocultured with MSCs for 48 h, IM was added for another 24 h. The final concentration of IM was 200 nm, 1 μm, and 5 μm. D, proliferation of K562/BV173 cells cocultured with different MSCs after NI treatment. The ratio of MSCs and K562/BV173 cells was 1:1. After K562/BV173 cells were cocultured with MSCs for 48 h, NI was added for another 24 h. The final concentration of NI was 30, 100, and 300 nm. E, viability of K562 cells after IM treatment in the absence or presence of different MSCs, F, viability of K562 cells after NI treatment in the absence or presence of different MSCs. G, representative FACS plot for annexin-V/PI-positive K562/BV173 cells cocultured with different MSCs after TKI treatment. *, p < 0.05 Student's t test.

Figure 3.

RMSCs mediate BCR/ABL-independent activation of STAT5 in K562 cells after NI treatment. K562 cells were cultured alone or with different MSCs at the indicated conditions (with 1 μm NI) for 24 h. RMSCs induce BCR/ABL-independent activation of STAT5. Cell lysates were probed with antibodies to phosphorylated BCR/ABL kinase (p-BCR/ABL), phosphorylated CrkL (p-CrkL), phosphorylated STAT5 Tyr-694, and β-ACTIN in Western blotting. *, p < 0.05 Student's t test.

Figure 4.

IL-7 mediates IM and NI resistance. A, IL-7 levels in different bone marrow samples. B, IL-7 levels in conditioned medium produced by different MSCs. C, detection of expression of IL-7 in mononuclear cells of bone marrow. D, proliferation of K562 cells after TKI treatment in the presence of RMSCs with or without anti-IL-7. E, proliferation of K562 cells after TKI treatment in the presence of RMSCs with or without siIL-7. F, representative FACS plot for propidium iodide-gated annexin-positive K562 cells after TKI treatment in the presence of RMSCs with or without anti-IL-7. G, representative FACS plot for propidium iodide-gated annexin-positive K562 cells after TKIs treatment in the presence of RMSCs with or without SiIL-7. *, p < 0.05 Student's t test.

Figure 5.

JAK1/STAT5 signaling pathway was activated by IL-7 in K562 and BV173 cells. K562 and BV173 cells were cultured alone or with different MSCs at the indicated conditions (with 1 μm IM) for 24 h. Cell lysates were probed with antibodies to JAK1, pJAK1, JAK3, pJAK3, STAT5, pSTAT5, PI3K, P-AKT, and β-ACTIN in Western blotting. A, representative blots and densitometry analysis of JAK1, pJAK1, JAK3, pJAK3, STAT5, pSTAT5, and β-ACTIN in K562 and BV173 cells after knockout of the IL-7 gene. B, representative blots and densitometry analysis of JAK1, pJAK1, STAT5, pSTAT5, and β-ACTIN in K562 and BV173 cells after knockout of the JAK1 gene. C, representative blots and densitometry analysis of PI3K, pAKT, and β-ACTIN in K562 cells. Results shown are representative of three independent experiments. β-ACTIN was considered as loading control. D, viability of K562 cells after IL-7 antibody and pimozide treatment. *, p < 0.05 Student's t test.

Figure 6.

RMSCs attenuated K562 xenograft growth in vivo. A, representative example of mice after transplantation with K562 cells alone, K562 + NCMSCs, K562 + SMSCs, K562 + RMSCs, and K562 + RMSCs + IL-7 siRNA by oral gavage for 40 days. B, representative photographs and mean weights of the isolated tumors on day 40 after implantation. Data are presented as mean ± S.E. of the mean. C, tumor growth curves.

Figure 7.

Increased expressions of pJAK1 and pSTAT5 were confirmed by immunofluorescence staining and Western blotting in tumor tissue. A, expression of pJAK1 and pSTAT5 in tumor tissue by immunofluorescence staining. B, representative blots and densitometry analysis of JAK1, pJAK1, STAT5, pSTAT5, and β-ACTIN in tumor tissue. DAPI, 4′,6-diamidino-2-phenylindole.