Activity of PIM Kinases in Chronic Lymphocytic Leukemia Modulates Tumor Cell Survival and Stromal Interactions through a Pleiotropic Mechanism Involving Modulation of CXCR4 - mTOR Pathway

The survival of chronic lymphocytic leukemia (CLL) cells depends on their interactions with microenvironment components, such as stromal and T cells. Lymph node microenvironment provides protective signals that enable the formation of proliferation centers and favor resistance to conventional chemotherapeutics. One of the key molecules engaged in the communication of CLL cells with their microenvironment is C-X-C chemokine receptor type 4 (CXCR4). The surface expression of CXCR4 is regulated by PIM1 (provirus integration site for Moloney murine leukemia virus) kinase. PIM 1-3 kinases are overexpressed in CLL cells and recent data suggest that targeting PIMs might be a rational therapeutic approach in this type of leukemia. Herein, we assessed associations of PIM kinase expression with clinical characteristics of CLL patients and investigated the consequences of PIM kinase inhibition for cell survival and CXCR4 - dependent signal transduction and migration of primary CLL cells.

In primary CLL cells from peripheral blood, PIM2 transcript abundance was higher than PIM1 and PIM3 (p<0.001). PIM2 expression was higher in patients with advanced disease (Rai 3-4, p=0.004). Higher PIM2 expression was also observed in patients who relapsed after first line treatment (p=0.005). Expression of PIM2 and PIM3 kinases in lymph nodes was significantly higher than in peripheral blood (p=0.024 and p=0.0059, respectively; Herishanu et al., 2010), suggesting a relationship between PIM kinase expression/activity and CLL cell microenvironment. To further explore the role of PIM kinases in these processes, we assessed whether PIM inhibition interferes with CXCR4 - mediated signaling and migration. We incubated primary CLL cells with a novel pan-PIM inhibitor SEL24-B489 and found decreased phospho-CXCR4 (Ser339) level and decreased CXCR4 surface expression. Primary CLL cells incubated with with SDF1 (500 ng/ml, 15-60 min) exhibited highly increased phosphorylations of mTOR (Ser2448) and Akt (Ser437). Since PIM kinases modulate mTOR signaling, we further investigated whether inhibition of PIM kinases with SEL24-B489 interferes with CXCR4/mTOR pathway. SEL24-B489 blocked baseline phosphorylation level of  mTOR negative regulator p-TSC2 (Ser1798).  Since TSC2 Ser1798 phosphorylation relieves its suppression on RHEB and promotes mTOR activity, we next assessed PIM inhibitor - induced changes in p-mTOR (Ser2448). Upon SEL24-B489 treatment, mTOR Ser2448 phosphorylation and activity of mTOR downstream substrates (p-Akt Ser473 and p-4EBP1 Thr37/46) markedly decreased. Pre-incubation of CLL cells with 10 uM SEL24-B489 or 10 uM mTOR inhibitor OSI-027 before SDF1 treatment restrained the increase of p-mTOR (Ser2448), p-Akt (Ser473), p-4EBP1 (Thr37/46) and p-TSC2 (Ser1798), and consequently impaired CLL cells migration in the SDF1 gradient. In 4 out of 7 analyzed patients the effect of SEL24-B489 on CLL migration was stronger than the effect of OSI-027 and in the remaining 3 patients, the effects of both inhibitors were similar. Since interactions of CLL cells with their microenvironment block the cytotoxicity of chemotherapeutic agents, we next compared the apoptotic response to SEL24-B489 in CLL cells cultured in the absence or presence of human bone marrow HS5 cells. CLL cells were seeded on HS5 monolayers and treated with SEL24-B489 (5 uM and 10 uM) for 48 hours. In 6 out of 7 cases SEL24-B489 overcame the protective signals from HS5 cells and induced apoptosis, although the cytotoxic effect of PIM inhibitor was stronger in the absence of stromal cells.

Taken together, our data demonstrate that CXCR4/SDF1 signal in chronic lymphocytic leukemia cells is transduced through mTOR pathway and that CXCR4 - triggered mTOR activity is modulated by PIM kinases. Pan-PIM inhibitor SEL24-B489 decreased CXCR4 surface expression and SDF-1 - triggered mTOR activity. Finally, SEL24-B489 decreased protective effects of tumor microenvironment and induced CLL cells apoptosis even in the presence of stromal cells. Since overexpression of PIM kinases might be associated with adverse clinical characteristics at diagnosis, PIM inhibition might be a rational therapeutic strategy in CLL.