Contributions
Abstract: PB1679
Type: Publication Only
Background
Acute myeloid leukemia (AML) is a disease characterized by an increase in immature myeloid blasts in the bone marrow (BM) as a consequence of the loss of normal differentiation and uncontrolled proliferation of malignant hematopoietic stem and progenitor cells (HSPCs). As for normal HSPCs, the interaction with the surrounding BM microenvironment, e.g. with mesenchymal stromal cells (MSCs), mediated by cell surface proteins is important for leukemic cell fate and characteristics such as proliferation, survival and drug resistance. Amongst them, Notch pathways play an important role by regulating the crosstalk between leukemia cells and stromal microenvironment (Seke et al. 2012). Recently, we described the HSPC supportive effect of expression of Notch ligand Jagged-1 (Jag1) in MSCs (Duryagina et al. 2013).
Aims
Here, we aim to investigate specific effects of Jag1 expression in MSCs on leukemia cells.
Methods
MSCs were isolated by density gradient centrifugation and plastic adherence of mononuclear cells from healthy donors and AML patients (n=10) and were characterized by flow cytometry, Western blot and ELISA. The hTert immortalized MSC line SCP-1 with Jag1 over-expression or expression of dominant negative mastermind 1 (dnMAML1), in which Jag1 signaling of MSCs is blocked, has been described recently (Duryagina et al. 2013). AML cell lines MV4-11 (FLT-ITD positive) or OCI-AML3 (FLT3 wildtype) were co-cultured with the different SCP-1 cells and were analyzed by proliferation, adhesion and survival assays as well as by quantitative real-time PCR.
Results
MSCs of AML patients expressed common cell surface molecules, such as CD73, CD90, CD105, CD44, CD146 and CD166 but with higher variations than normal MSCs. Expression of the Notch ligand Jag1 was decreased in AML MSCs (detected in 3 out of 10 samples). The proliferation and adhesion capacity of MV4-11 cells was clearly decreased to about 35% on a SCP-1/Jag1 monolayer in comparison to wildtype and dnMAML1 cells. Addition of the g-secretase inhibitor DAPT partly rescued this effect confirming an involvement of Notch signaling. For OCI-AML3 cells no difference in proliferation and adhesion could be detected on the different SCP-1 layers. Treatment of MV4-11 cells with the tyrosine kinase inhibitor PKC412 (Midostaurin) or Ara-C increased the number of apoptotic and dead cells as detected by Annexin-V/PI staining in co-culture with SCP-1/Jag1 cells, suggesting that Jag1 over-expression can reverse the niche protective effect at least in part. Co-culture of MV4-11 cells on SCP-1/Jag1 cells caused a stronger induction of the Notch signaling pathway as shown by higher Hes-1 (20-fold) or Hey-1 (15-fold) mRNA expression levels than co-culture with SCP wildtype cells, whereas in OCI-AML3 this increase was significantly lower (2.5- and 1.5-fold, respectively). Whereas co-culture of AML cells caused inhibited secretion of SDF-1 by stromal cells, the co-culture of AML cells on SCP-1/Jag1 cells caused increased expression of CXCR4 mRNA in leukemia cells and accordingly 2- to 3-fold higher SDF-1 expression and secretion in the stromal cells in comparison to SCP-1 wildtype co-cultures. Again, these effects were much weaker in OCI-AML3 cells suggesting a role of FLT3 mutation.
Conclusion
In summary, we have demonstrated that the Jag1/Notch/Hes-Hey1 axis cause tumor inhibiting effects in FLT3 mutated AML cells. The different effects of Jag1 expression in MSCs on leukemia and normal hematopoietic cells may serve as prognostic marker and open a new therapeutic window.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia, Flt3-ITD, Mesenchymal cells
Abstract: PB1679
Type: Publication Only
Background
Acute myeloid leukemia (AML) is a disease characterized by an increase in immature myeloid blasts in the bone marrow (BM) as a consequence of the loss of normal differentiation and uncontrolled proliferation of malignant hematopoietic stem and progenitor cells (HSPCs). As for normal HSPCs, the interaction with the surrounding BM microenvironment, e.g. with mesenchymal stromal cells (MSCs), mediated by cell surface proteins is important for leukemic cell fate and characteristics such as proliferation, survival and drug resistance. Amongst them, Notch pathways play an important role by regulating the crosstalk between leukemia cells and stromal microenvironment (Seke et al. 2012). Recently, we described the HSPC supportive effect of expression of Notch ligand Jagged-1 (Jag1) in MSCs (Duryagina et al. 2013).
Aims
Here, we aim to investigate specific effects of Jag1 expression in MSCs on leukemia cells.
Methods
MSCs were isolated by density gradient centrifugation and plastic adherence of mononuclear cells from healthy donors and AML patients (n=10) and were characterized by flow cytometry, Western blot and ELISA. The hTert immortalized MSC line SCP-1 with Jag1 over-expression or expression of dominant negative mastermind 1 (dnMAML1), in which Jag1 signaling of MSCs is blocked, has been described recently (Duryagina et al. 2013). AML cell lines MV4-11 (FLT-ITD positive) or OCI-AML3 (FLT3 wildtype) were co-cultured with the different SCP-1 cells and were analyzed by proliferation, adhesion and survival assays as well as by quantitative real-time PCR.
Results
MSCs of AML patients expressed common cell surface molecules, such as CD73, CD90, CD105, CD44, CD146 and CD166 but with higher variations than normal MSCs. Expression of the Notch ligand Jag1 was decreased in AML MSCs (detected in 3 out of 10 samples). The proliferation and adhesion capacity of MV4-11 cells was clearly decreased to about 35% on a SCP-1/Jag1 monolayer in comparison to wildtype and dnMAML1 cells. Addition of the g-secretase inhibitor DAPT partly rescued this effect confirming an involvement of Notch signaling. For OCI-AML3 cells no difference in proliferation and adhesion could be detected on the different SCP-1 layers. Treatment of MV4-11 cells with the tyrosine kinase inhibitor PKC412 (Midostaurin) or Ara-C increased the number of apoptotic and dead cells as detected by Annexin-V/PI staining in co-culture with SCP-1/Jag1 cells, suggesting that Jag1 over-expression can reverse the niche protective effect at least in part. Co-culture of MV4-11 cells on SCP-1/Jag1 cells caused a stronger induction of the Notch signaling pathway as shown by higher Hes-1 (20-fold) or Hey-1 (15-fold) mRNA expression levels than co-culture with SCP wildtype cells, whereas in OCI-AML3 this increase was significantly lower (2.5- and 1.5-fold, respectively). Whereas co-culture of AML cells caused inhibited secretion of SDF-1 by stromal cells, the co-culture of AML cells on SCP-1/Jag1 cells caused increased expression of CXCR4 mRNA in leukemia cells and accordingly 2- to 3-fold higher SDF-1 expression and secretion in the stromal cells in comparison to SCP-1 wildtype co-cultures. Again, these effects were much weaker in OCI-AML3 cells suggesting a role of FLT3 mutation.
Conclusion
In summary, we have demonstrated that the Jag1/Notch/Hes-Hey1 axis cause tumor inhibiting effects in FLT3 mutated AML cells. The different effects of Jag1 expression in MSCs on leukemia and normal hematopoietic cells may serve as prognostic marker and open a new therapeutic window.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia, Flt3-ITD, Mesenchymal cells