Sphingosine kinase 1 participates in the activation, proliferation and survival of chronic lymphocytic leukemia cells

GIORDANO MIRTA; GAMBERALE ROMINA; BURGOS RUBEN; SÁNCHEZ-ÁVALOS JULIO CÉSAR; FERNÁNDEZ GRECCO HORACIO; BEZARES RAIMUNDO F; STANGANELLI CARMEN; SLAVUTSKY IRMA; PODAZA ENRIQUE; RISNIK DENISE; BORGE MERCEDES; COLADO ANA; GIORDANO MIRTA; GAMBERALE ROMINA; BURGOS RUBEN; SÁNCHEZ-ÁVALOS JULIO CÉSAR; FERNÁNDEZ GRECCO HORACIO; BEZARES RAIMUNDO F; STANGANELLI CARMEN; SLAVUTSKY IRMA; PODAZA ENRIQUE; RISNIK DENISE; BORGE MERCEDES; COLADO ANA; OPPEZZO PABLO; CRANCO SANTIAGO; CABREJO MARÍA; DE BRASI CARLOS DANIEL; ELÍAS ESTEBAN ENRIQUE; ALMEJÚN MARÍA BELÉN; OPPEZZO PABLO; CRANCO SANTIAGO; CABREJO MARÍA; DE BRASI CARLOS DANIEL; ELÍAS ESTEBAN ENRIQUE; ALMEJÚN MARÍA BELÉN

HAEMATOLOGICA

FERRATA STORTI FOUNDATION

Año: 2017 vol. 102 p. 257 - 260

0390-6078

Sphingosine kinases (SKs) have received the most attention as important enzymes in cancer biology. They participate in the regulation of bioactive sphingolipid metabolism by producing sphingosine-1 phosphate (S1P) which mediates several biological functions, including cell growth, differentiation, cell survival, migration, and angiogenesis among others1. S1P generation depends on the conversion of sphingosine to S1P, in a reaction catalyzed by two isoforms of SKs, SK1 and SK2, and its levels are tightly controlled via a rapid degradation by intracellular S1P lyases (S1PL) or dephosphorylated by S1P phosphatases1. Once produced, S1P may function as an intracellular second messenger and/or can be exported outside the cells, where it binds to specific S1P receptors (S1PRs) and initiates downstream signaling pathways, in a paracrine or autocrine manner, in a process known as ?inside-out? signaling1. Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized by the accumulation of clonal B lymphocytes in peripheral blood and lymphoid tissues. Given that several studies have implicated the SKs/S1P/S1PL pathway as an essential regulator of cell proliferation and survival in cancer cells2-4 we evaluated the role of SKs and S1PL in leukemic cells from CLL patients. To this aim we first measured the basal expression of SKs mRNAs and S1PL mRNA by qRT-PCR on purified B cells from CLL patients and age-matched healthy donors. We found that CLL cells expressed heterogeneous levels of SK1, SK2 and S1PL mRNA, while only SK1 mRNA was statistically significant higher compared to healthy donors (Figure 1A). As we observed at mRNA level, when SK1 was evaluated by western blot we found a higher expression of the protein in B cells from CLL samples compared to healthy donors (Figure 1B and supplementary Figure S1A). In addition, within CLL patients, there was a positive correlation between SK1 mRNA and protein expression (Supplementary Figure S1B). We also observed that the ratio between SK1 and S1PL were increased for B cells from CLL patients compared to B cells from healthy donors (Figure 1C), while no statistically significant differences were found for SK2/S1PL ratios (Supplementary Figure S1C). Remarkably, CLL patients with higher SK1/S1PL ratios preferentially belonged to Binet B or C clinical stages, are unmutated, CD38+, CD49d+ and showed a progressive disease (Table 1 and Supplementary Table S1), suggesting that SK1/S1PL molecules might participate in the clinical outcome of the patients favoring the progression of the disease. Malignant B cells from CLL patients mainly receive advantageous signals in lymphoid tissues, where the supportive microenvironment and B cell receptor (BCR) signaling promote their activation and proliferation, modulate cell adhesion and migration and protect CLL cells from spontaneous and drug-induced apoptosis5. We have previously reported that CLL cells activated by signals from the supportive microenvironment transiently impair the expression of S1PR16, in a process that may extend the residency of the leukemic clone within the survival niches of lymphoid tissues. We here wanted to determine whether the activation of CLL cells has any influence on SK1/S1PL ratios. To this aim, purified B cells from CLL patients were cultured in the presence of immobilized anti-IgM mAbs and CD40L, CpG or HS5 cell line as a feeder layer and, as expected, after 24 hours of culture, the stimuli induced the up-regulation of the activation marker CD69 on CLL cells (not shown). Interestingly, we found that CLL activation enhanced SK1/S1PL ratios in all of the patients evaluated (Figure 1D), independently of their clinical stage or the prognosis group (not shown). Ibrutinib, by impairing leukemic cell activation (not shown) was able to strongly reduce the up-regulation of SK1/S1PL ratios induced by the stimulus (Supplementary Figure S2). Moreover, in order to evaluate the expression of SK1/S1PL ratios within in vivo activated CLL cell subpopulations, we took advantage of the fact that we had previously obtained mRNA samples of in vivo activated CLL cells subpopulations and non-activated counterpart of the same patient6. When SK1, SK2 and S1PL mRNA levels were evaluated in the proliferative fraction of circulating CLL cells (PF, IgG+ cells) described by Palacios et al7 and the quiescent fraction from the same patient (IgM+IgG- cells, QF), we found higher SK1/S1PL ratios in the PF of all the three CLL patients evaluated (Figure 1E). Additionally, bone marrow leukemic cells expressing high levels of CD38, which defines a subpopulation of activated lymphocytes, showed higher SK1/S1PL ratios compared to CD38 low or negative counterparts (n=3) (Figure 1F), indicating that in vivo activated CLL cells expressed higher ratios of SK1/S1PL compared with the rest of the leukemic clone.Then, to test whether the inhibition of SKs could modify the survival of CLL cells, we employed SKI-II, which is an orally bioavailable inhibitor for SK1 and SK2 that blocks S1P production and cell proliferation8. When peripheral blood mononuclear (PBMC) cells from CLL patients were cultured with different doses of SKI-II, we found that SKI-II induced cell death in a dose-dependent way (Figure 2A and Supplementary Figure S3A). We observed that the percentage of cell death induced by 50μM of SKI-II inversely correlated with the basal SK1/S1PL ratios of each sample (Supplementary Figure S3B). In line with this, CLL patients with low SK1/S1PL ratios were more sensitive to 50 μM of SKI-II compared to patients with high SK1/S1PL ratios (Figure 2B). In addition, we found that non-apoptotic doses of SKI-II (15 μM) increased the susceptibility of CLL cells to die by other therapeutic agents, such as fludarabine, bendamustine and ibrutinib (Figure 2C), more markedly in CLL patients with low SK1/S1PL ratios compared to CLL patients with high SK1/S1PL ratios (supplementary figure S3C). Similar results were recently reported by others showing that SKs inhibition induces apoptosis in primary multiple myeloma9, acute myeloid leukemia10 and natural killer-large granular lymphocyte leukemia cells11 and also sensitizes CLL cells to other therapeutic agents such as EGCG (-epigallocatechin-O-3-gallate)12 or lysosome-targeting drugs13. SKI-II induced cell death was associated with a downregulation of BCL-2 expression in CLL cells (Supplementary Figure S3D), as previously reported in SKI-II treated- gastric cancer cells14. Thus, CLL samples with low SK1/S1PL ratios, which were more sensitive to 50 μM of SKI-II (Figure 2B), showed a higher BCL-2 downregulation upon SKI-II treatment compared to CLL patients with high SK1/S1PL ratios (Figure 2D and supplementary Figure S3E). On the other hand, we found that non-apoptotic doses of SKI-II were able to impair CLL activation induced by anti-IgM+CD40L at 24 hours, measured by the upregulation of CD69 (Figure 2E), and also CLL proliferation evaluated at 96 hours (Figure 2F). Remarkably, those samples with high SK1/S1PL ratios, presented a greater leukemic cell proliferation induced by anti-IgM+CD40L compared to patients with low SK1/S1PL (Figure 2F), suggesting that SK1/S1P/S1PL pathway might regulate this proliferative response in CLL. Representative CFSE histograms are depicted in Supplementary Figure S3F. Finally, in order to determine whether S1PRs signaling may counteract the effects of SKI-II, we added exogenous S1P to the cultures and found that it rescued CLL cells from the cell death induced by 50 μM of SKI-II (Figure 2G) and from the anti-proliferative effect of 15 μM of SKI-II (Figure 2H). These findings suggest that S1P production by CLL cells may favor leukemic cell survival and proliferation in an ?inside-out? signaling manner acting through S1PRs1.Altogether, the results presented here and our previous data showing that the activation of CLL cells transiently impair the expression of S1PR16, allow us to hypothesize about the role of SK1/S1P/S1PL axis in CLL. As shown in the model of Supplementary Figure S4 the possibility exists that microenvironment signals from lymphoid tissues, by increasing SK1/S1PL ratios in leukemic CLL cells, favors S1P production. Exported S1P can then bind S1PRs, reduce S1PR1 expression6 and transduce stimulating signals to the leukemic clone.In conclusion, our results suggest that SK/S1P/S1PL pathway supports the survival and proliferation of CLL cells favoring the progression of the disease and encourage the use of SKs inhibitors in combined therapy as a promising treatment option in the future.