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P.S. Godavarthy et al.
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Figure 6. BCR-ABL1 modulates SCL/TAL1-binding to the CD44 regulatory element via pAKT. (A) Immunoblot of lysates of K562 cells treated with vehicle, imatinib (10 mM), the PI3-kinase inhibitor wortmannin (20 mM) or the AKT-inhibitor MK-2206 (20 mM) and probed with antibodies to SCL/TAL1, pSCL/TAL1 T90, pSCL/TAL1S122, AKT, pAKT and lamin. Molecular weights are as indicated. The immunoblot is representative of three independent experiments. (B-D) Quantitative mass spectrometry (MS) of K562 cells treated with vehicle (dimethylsulfoxide, DMSO), imatinib or the AKT inhibitor MK-2206 for 0, 2 or 4 h using triple stable isotope labeling with amino acids in cell culture (SILAC) and light-, medium- or heavy-labeled cells. After treatment, differently labeled cells were mixed and followed by MS analysis. (B) An averaged MS1 spec- trum showing the SILAC triplets of the pS122-bearing peptide “oxMVQLpSPPALAAPAAPGR” on SCL/TAL1 in AKT-inhibited cells. The arrows indicate the mass increments resulting from the medium (+6 Da) or heavy (+10 Da) SILAC-labeling on arginine. The relative intensities of the SILAC triplets revealed the changes of phosphorylation level on the peptide during the course of AKT inhibition. (C, D) Areas under the extracted ion chromatogram (XIC) after MS by triple SILAC labeling of the cells in (B) showing the relative abundance of pSCL/TAL1 S122 after inhibition with imatinib (10 mM) (C) and the AKT inhibitor MK-2206 (20 mM) (D).
sion of SCL/TAL1 and CD44 in CD34+-sorted samples from chronic phase CML patients (n=59). We observed a statistically significant negative correlation (P=0.002, cor- relation coefficient -0.40) between SCL/TAL1 and CD44 expression in these samples (Figure 7E). Lastly, we ex- amined the association between SCL/TAL1 expression and outcomes after allogeneic transplantation in chronic phase CML patients (n=37),35 although this procedure is no longer the standard of care in CML. When dichotomi- zing SCL/TAL1 into high and low expression groups at the third quartile, we found that low SCL/TAL1 expression was statistically significantly associated with increased risk of relapse (P=0.033) (Figure 7F) and inferior relapse- free survival (P=0.024) (Figure 7G). In summary, these data suggest that expression of SCL/TAL1 (and, therefore, con- verse expression of CD44) may correlate with disease stage and survival in human CML patients, although larger cohorts would be needed to prove this definitively.
Discussion
In this study we show that inhibition of binding of BCR-ABL1+ cells to E-selectin in the vascular niche increa- ses cell cycle progression and response to imatinib thera- py. Furthermore, our data also imply that SCL/TAL1 is a regulator of the expression of CD44, whereby SCL/TAL1 is activated by AKT downstream of BCR-ABL1 (Online Supplementary Figure S12). In turn, CD44 influences the cell cycle of BCR-ABL1+ cells via its binding to E-selectin. The data connect the previously unknown mechanism of increased expression of CD44, a ligand for E-selectin and cancer stem cell marker, on BCR-ABL1+ cells with tran- scriptional regulation by SCL/TAL1, E-selectin in the vas- cular niche, engraftment in the BM microenvironment, cell cycle progression and response to therapy.
Our data on CML LSC contrast with findings in the normal HSC niche, in which a prominent role for E-
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