Sphingosine Kinases (SK): Key Molecules Associated with the Activation, Proliferation and Ibrutinib-Induced Cell Death of Chronic Lympocytic Leukemia Cells
Authors: Almejun Maria Belen , Borge Mercedes , Colado Ana , Podaza Enrique , Risnik Denise , De Brasi Carlos Daniel , Oppezzo Pablo , Cabrejo Maria , Fernandez Grecco Horacio , Bezares Raimundo F. , Cranco Santiago C , Burgos Ruben A. , Sanchez Avalos Julio Cesar , Giordano Mirta N. , Gamberale Romina 
Published: October 2019

Abstract
Leukemic cells from CLL patients can survive and proliferate within lymphoid tissues where the supportive microenvironment favors their accumulation. We have previously reported that the activation of CLL cells reduces the expression of the main receptor for sphingosine 1-phosphate (S1P) (Borge M, J Immunol, 2014), a bioactive phospholipid that participates in lymphocyte egress from lymphoid tissues. S1P also mediates several biological functions, including cell growth stimulation and protection of apoptosis, through receptor independent intracellular mechanisms. S1P is generated by two isoforms of sphingosine kinases (SK1/2) and its levels are tightly controlled via degradation by intracellular S1P lyases (S1PL). Several studies have implicated the SK/S1P/S1PL pathway as an essential regulator of cell proliferation and survival in different cancer cells. The aims of our study were: a) to evaluate the expression of SK and S1PL in CLL cells, b) to assess whether key microenvironment signals are able to modulate this expression and c) to evaluate the effect of SK inhibitors on the activation and survival of the leukemic clone.
We measured the basal expression of SK and S1PL by qRT-PCR on purified B cells from CLL patients (n=22) and aged-matched healthy donors (n=9), and found that CLL cells express high levels of SK1, favoring an increased SK1/S1PL ratio in the malignant clone compared to healthy B lymphocytes ( p<0.05). Similar results were obtained when SK2 was evaluated. The in vitro activation of CLL cells with anti-IgM+CD40L increased SK1/S1PL ratio (n=10, p<0.01) and the expression of the activation marker CD69. To evaluate the expression of SK1/S1PL within in vivo activated CLL cell subpopulations, we segregated the proliferative fraction (PF) of circulating CLL cells from the quiescent fraction (QF) in the same sample. As it was previously described, this PF is characterized by the presence of an active class-switch recombination process and a high expression of proliferation-related genes, such as Ki-67, c-myc, CD49d, and p27-Kip1 (Palacios F, Blood, 2010). Interestingly, SK1/S1PL ratio was increased in the PF compared with the QF (n=3). Additionally, bone marrow leukemic cells expressing high levels of CD38, which defines a subpopulation of activated lymphocytes, showed a higher S1P/S1PL ratio compared to CD38 low or negative counterparts (n=3),showing that the in vivo activated CLL cells expressed higher ratios of SK1/S1PL compared with the rest of the leukemic clone. Finally, we wondered whether the inhibition of SK impairs the survival, activation and proliferation of the leukemic clone. To this aim we employed a commercial selective SK1 and SK2 inhibitor (SKI-II, 5 and 15μM), which did not affect cell viability (n=10, evaluated at 24, 48, 72 and 96hs). However, it was able to impair the expected upregulation of CD69 induced by IgM+CD40L at 24hs (n=10, p<0.001) and the leukemic cell proliferation evaluated by CFSE dilution assay at 96hs (n=10 , p<0.001). Moreover, while SKI-II did not increase the sensitivity of CLL cells to Fludarabine or Bendamustine, it was able to enhance the cell death induced by Ibrutinib (0.3 and 3μM) (n=5, p<0.05 and p<0.01 respectively).
Taking together, our results suggest that SK/S1P/S1PL axis might participate in the accumulation of the malignant clone in CLL patients and the disruption of this pathway might be a potentially effective treatment option in the future.

Disclosures
Bezares: Janssen: Honoraria. Gamberale:Janssen: Honoraria.