Phosphorylation of Spleen Tyrosine Kinase (Syk) Appears to Be Related to the Blast Phase of Chronic Myeloid Leukemia
Authors: Bourgne Céline , Janel Alexandre , Chassagne Jacques , Rapatel Chantal , Tournilhac Olivier , Guerci Agnès , Lioret Frédérique , Briançon Aurélie , Berger Juliette , Bamdad Mahchid , Boiret-Dupré Nathalie , Berger Marc G 
Published: October 2019

Abstract
Abstract 4425

Introduction:
Despite the major benefit of TKI in the treatment of Chronic Myeloid Leukemia (CML) chronic phase, some of patients are resistant or progress to blast phase (BP) becoming not very accessible to therapy. We have been interested in the Syk molecule as a potential marker for CML progression for several reasons: i) its potential interaction with Src kinases, activated by BCR-ABL, and tyrosine kinase receptors, ii) its involvement in the molecular complexes activating actin and the cytoskeleton and integrin signalling pathways, regulating cell adhesion, a property that is impaired in CML, iii) its interaction with the PI3K/Akt pathway, activated by BCR-ABL. Furthermore, resistance to nilotinib was recently showed dependent on Syk expression.

Method:
The amount of Syk transcript was analyzed in primary cells using the 2-ΔΔ Ct method and was normalized to the endogenous reference gene (β2-microglobuline) and K562 cells as the calibrator. Using flow cytometry, we evaluated the expression of Syk and pSyk348 in K562 cells and in polymorphonuclear cells from 3 healthy donors (HD-PMN), primitive CML cells from 15 patients in chronic phase (CP) (patients #1 to #15) at diagnosis, in the blast cells from 4 patients in accelerated phase (AP) (patient#16, #17, #18 and #19) and from 2 patients in blast crisis (BC) (patient#20, #21). The level of intracellular dasatinib (DAS) was evaluated by an original flow cytometry method (Bourgne et al. Cytometry Part A, in press).

Results:
We observed a significant over expression of Syk mRNA in BP-CML cells, whereas there is no difference between HD-PMN and CP-CML cells. At the protein level we detected a decrease (2 times; p<0.001) in and a tendency to increase (1.5 times; p=0.5) in the expression of Syk in CP-CML cells and BP-CML cells respectively compared with HD-PMN cells. Interestingly, we did not observe any expression of pSyk348 in HD-PMN or in granulocytic cells from CP-CML (n=15) but we systematically detected pSyk348 expression in the blast cells of the 6 patients (positive/control ratio: 2.3 ± 0.3) in advanced phases of CML. Moreover we did not found Syk phosphorylation in CP cells from one patient resistant to imatinib, then nilotinib and dasatinib but we detected pSyk348 only when his CML progressed, strengthening the hypothesis of a link between Syk phosphorylation and BP of CML. We confirmed in vitro that Dasatinib was able to rapidly (15 min) inhibit Syk phosphorylation in K562 cells and in blast cells from patient #17. Then we could follow dasatinib uptake into target blast cells, and expression of pSyk348 before and 6, 12, 36, 60, 84 and 136 hours after the first dose of DAS. We observed a significant storage of DAS in blast cells reaching a plateau after the 4th dose even though analyses were done 12 hrs after each dose. However, after a slight fall of blood leukocytes and blast cells numbers corresponding to a sharp drop of pSyk348 we observed an increase of blood malignant cells in parallel of a strong recurrence of pSyk348 at H60.

Discussion:
We observed a constitutive expression of Syk348 only in blast cells from advanced phases of CML, including in one patient we could follow from the TKI resistant phases to blast phase, strongly suggesting that Syk activation could be a pertinent biomarker for CML progression and could represent a potential target for combinatory therapy. The fact that we observed in one patient a correlation between Syk348 expression and malignant cell resistance even though cells stored dasatinib suggests a BCR-ABL/Src kinases independent mechanism of Syk phosphorylation in blast cells. Targeting Syk in BP-CML should offer new therapeutic option to patients with disease progression.

Disclosures:
No relevant conflicts of interest to declare.