Sphingosine Kinases (SK): Key Molecules Associated with the Activation, Proliferation and Ibrutinib-Induced Cell Death of Chronic Lympocytic Leukemia Cells.

ALMEJUN MB; BORGE M; COLADO A, PODAZA E, RISNIK D,; DE BRASI CD; PABLO OPPEZZO; CABREJO M, FERNANDEZ GRECCO H, BEZARES RF, CRANCO SC, BURGOS RA, SANCHEZ AVALOS JC,; GIORDANO MN; GAMBERALE R

Orlando

Congreso; 57th Annual Meeting & Exposition Congress of the American Society of Hematology (ASH); 2015

American Society of Hematology (ASH)

Leukemic cells from CLL patients cansurvive and proliferate within lymphoid tissues where the supportivemicroenvironment favors their accumulation. We have previously reported thatthe activation of CLL cells reduces the expression of the main receptor for sphingosine1-phosphate (S1P) (Borge M, J Immunol, 2014), a bioactive phospholipid thatparticipates in lymphocyte egress from lymphoid tissues. S1P also mediatesseveral biological functions, including cell growth stimulation and protectionof apoptosis, through receptor independent intracellular mechanisms. S1P isgenerated by two isoforms of sphingosine kinases (SK1/2) and its levels aretightly controlled via degradation by intracellular S1P lyases (S1PL). Several studieshave implicated the SK/S1P/S1PL pathway as an essential regulator of cellproliferation and survival in different cancer cells. Theaims of our study were: a) to evaluate the expression of SK and S1PL in CLLcells, b) to assess whether key microenvironment signals are able to modulatethis expression and c) to evaluate the effect of SK inhibitors on theactivation and survival of the leukemic clone.Wemeasured the basal expression of SK and S1PL by qRT-PCR on purified B cellsfrom CLL patients (n=25) and aged-matched healthy donors (n=9), and found thatCLL cells express high levels of SK1, favoring an increased SK1/S1PL ratio inthe malignant clone compared to healthy B lymphocytes (p<0.005). Similarresults were obtained when SK2 was evaluated. The in vitro activation of CLL cells with anti-IgM+CD40L increasedSK1/S1PL ratio (n=8, p<0.01) and the expression of the activation markerCD69. To evaluate the expression of SK1/S1PL within in vivo activated CLL cell subpopulations, we segregated theproliferative fraction (PF) of circulating CLL cells from the quiescentfraction (QF) in the same sample. As it was previously described this PF ischaracterized by the presence of an active class-switch recombination processand 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, whichdefines a subpopulation of activated lymphocytes, showed a higher S1P/S1PLratio compared to CD38 low or negative counterparts (n=3), showing that the in vivo activated CLL cells expressedhigher 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 acommercial selective SK1 and SK2 inhibitor (SKI-II, 5 and 15μM), which did notaffect cell viability (n=15, evaluated at 24, 48, 72 and 96hs). However, it wasable to impair the expected upregulation of CD69 induced by IgM+CD40L at 24hs(n=10, p<0.05) and the leukemic cell proliferation evaluated by CFSEdilution assay at 96hs (n=10, p<0.01). Moreover, while SKI-II did notincrease the sensitivity of CLL cells to Fludarabine or Bendamustine (notshown), it was able to enhance the cell death induced by Ibrutinib (0.3 and3μM) (n=10, p<0.05).Taking together, our results suggest thatSK/S1P/S1PL axis might participate in the accumulation of the malignant clonein CLL patients and the disruption of this pathway might be a potentiallyeffective treatment option in the future.