Page 153 - 2020_01-Haematologica-web

The vascular niche in chronic myeloid leukemia
A
B
Figure 5. Expression of CD44 is influenced by BCR-ABL1. (A) Median fluorescence intensity (MFI) of CD44 on BaF3 cells transduced with empty vector or BCR-ABL1 plated on E- selectin after treatment with vehicle or 10 mM imatinib [P values as indicated, analysis of variance (ANOVA); Tukey test, n=3]. (B) MFI of CD44 (fold change) on the non- adherent and adherent frac- tions of BCR-ABL1+ BaF3 cells after treatment with vehicle, GMI-1271, imatinib or GMI- 1271 and imatinib, normalized to vehicle (P=0.005 for vehicle vs. GMI-1271 plus imatinib, ANOVA; Tukey test, n=4).
“oxMVQLpSPPALAAPAAPGR” of SCL/TAL1 in AKT- inhibited cells (Figure 6B and Online Supplementary Figure S11A). More precise quantitative measurements by com- paring the areas under the extracted ion chromatograms of individual SILAC triplets revealed the decrease of pTal1 S122 both 2 and 4 h after inhibition with imatinib (Figure 6C) or MK-2206 (Figure 6D). Taken together, these data suggest that SCL/TAL1 regulates the expression of CD44 via binding to the CD44 regulatory element in a BCR- ABL1- and AKT-dependent manner. BCR-ABL1 is known to activate AKT (Figure 6A),32,33 which phosphorylates SCL/TAL1 at S122 and T90, which in turn regulates the activity of the CD44 regulatory element by acting as a transcriptional repressor. Binding of SCL/TAL1 leads to decreased expression of CD44, decreased adhesion to the vascular niche in CML and an increase in cell cycling.
SCL/TAL1 influences outcome in human chronic myeloid leukemia
Given our observations in murine models and murine or human CML cells, we correlated levels of CD44 and SCL/TAL1 in leukocytes from healthy individuals and patients with CML. This revealed lower delta cycle threshold values for SCL/TAL1 and higher delta cycle threshold values for CD44 in healthy individuals (P<0.0001) (Figure 7A), while this relationship was reversed in human CML samples (P<0.0001) (Figure 7B), suggesting that SCL/TAL1 also acts as a transcriptional re- gulator in human CML cells. In order to test the signifi- cance of SCL/TAL1 and/or CD44 for phase of CML and disease course, we examined published datasets of SCL/TAL1 and CD44 gene expression in human CML samples34,35 and identified that SCL/TAL1 levels in patients in chronic phase (P=0.042) (Figure 7C) and – as a trend - accelerated phase were lower than in normal human CD34+ cells, as expected. CD44 expression increased with progression of CML from chronic to advanced phase CML with the highest CD44 expression found in samples from patients in blast crisis (P<0.0001) (Figure 7D).
As we did not establish a clear inverse relationship between SCL/TAL1 and CD44 expression in samples from individual patients from this dataset, we examined expres-
an increase in SCL/TAL1) is synergistically enhanced by cotreatment with imatinib.
CD44 expression influences adhesion and cell cycle status in imatinib-resistant chronic myeloid leukemia
Imatinib-resistance due to the point mutation BCR- ABL1T315I in CML is associated with worse clinical out- come28,29 and is due to altered interactions between BCR- ABL1T315I+ cells and the BM microenvironment.30 In support of this, the expression of CD44 was higher in BCR- ABL1T315I+ than in BCR-ABL1+ BaF3 cells (Online Supplementary Figure S10A, B) and also higher than in cells resistant to imatinib due to other point mutations (P=0.034) (Online Supplementary Figure S10B). Consistently, adhesion of BCR-ABL1T315I+ BaF3 cells to E-selectin was increased compared to that of BCR-ABL1+ cells (P=0.011) (Online Supplementary Figure S10C) and a larger percentage of adherent, but not non-adherent, BCR-ABL1T315I+ BaF3 cells were found in the G0 phase of the cell cycle (P=0.042) (Online Supplementary Figure S10D). These data support our findings on the role of CD44 in influencing the cell cycle in BCR-ABL1+ cells and suggest that increased CD44 expression and increased binding to E-selectin by BCR- ABL1T315I+ cells may contribute to dormancy and imatinib resistance.
BCR-ABL1 modulates SCL/TAL1-binding to the CD44 regulatory element via pAKT
As SCL/TAL1 activity is regulated by phosphorylation at S122 and T90, we hypothesized that a kinase down- stream of BCR-ABL1, such as AKT, may be phosphoryla- ting SCL/TAL1.31 In confirmation of this, we demonstra- ted that treatment of K562 cells with imatinib, the phos- phoinositide-3-kinase (PI3K) inhibitor wortmannin, and in particular the AKT inhibitor MK-2206 reduced the expres- sion of pTal1S122 and pTal1T90 (Figure 6A). Furthermore, quantitative mass spectrometry, performed using triple stable isotope labeling with amino acids in cell culture (SILAC)-labeling of K562 cells treated with vehicle, ima- tinib or MK-2206, demonstrated the mass increments resulting from medium (+6 Da) or heavy (+10 Da) SILAC- labeling on arginine on the pS122-bearing peptide
haematologica | 2020; 105(1)
143