Microenvironment-Induced Expression of PIM Kinases Supports Chronic Lymphocytic Leukemia Cells Survival and Promotes CXCR4-mTOR Pathway Dependent Migration
Authors: Bialopiotrowicz Emilia , Gorniak Patryk , Pula Bartosz , Noyszewska-Kania Monika , Makuch-Lasica Hanna , Nowak Grazyna , Szydlowski Maciej , Jablonska Ewa , Sewastianik Tomasz , Polak Anna , Lech-Maranda Ewa , Budziszewska Bozena Katarzyna , Wasylecka Maja , Borg Katarzyna , Warzocha Krzysztof , Czardybon Wojciech , Galezowski Michal , Windak Renata , Brzozka Krzysztof , Juszczynski Przemyslaw 
Published: October 2019

Abstract
Lymph node microenvironment provides chronic lymphocytic leukemia (CLL) cells with pro-survival and protective signals, fostering resistance to conventional chemotherapeutics. CLL cells overexpress oncogenic PIM kinases, which modulate proteins engaged in transcription, translation, apoptosis, cell cycle and adhesion/motility (Mol Cancer Ther 2014, 13: 1231-45). Herein, we searched for the link between tumor microenvironment and PIMs expression, compared the clinical characteristics of CLL patients with high versus low expression of PIM kinases, and investigated the consequences of their inhibition with newly developed pan-PIM inhibitor, SEL24-B489 in primary CLL cells.
We first evaluated the expression of PIM kinases in CD19+ cells derived from 88 newly diagnosed CLL cases. Patients with unmutated IGHV status exhibited significantly higher PIM1 transcript levels than patients with mutated IGHV genes. Subjects with advanced CLL (Binet C) exhibited higher PIM2 expression than patients in Binet A/B stage. Significantly higher PIM2 transcript abundance at the time of diagnosis 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, suggesting a relationship between PIM kinase expression/activity and CLL cell microenvironment. To further explore the role of microenvironment in the control of PIM expression, peripheral blood CLL cells were incubated with anti-IgM or CD40 ligand. Both stimuli induced PIM1 and PIM3 expression. Co-culture of CLL cells with stromal cell (HS5) monolayers promoted the expression of PIM3 isoform.
We next assessed the consequences of PIM inhibition in CLL cells using novel pan-PIM inhibitor, SEL24-B489. Incubation with SEL24-B489 decreased phosphorylation of PIM substrates, p-FOXO1/3a(T24/T32) and p-4EBP1(S65), and induced dose-dependent apoptosis in 27 out of 28 analyzed cases, regardless of the IGHV mutation status and including relapsed patients. Of note, SEL24-B489 induced higher apoptotic response in primary CLL cells than referential pan-PIM inhibitor AZD1208. CLL cells with 17p13 deletion and obtained from chemo-refractory patients were also vulnerable to SEL24-B489, suggesting that functional p53 is not required for execution of SEL24-B489-mediated apoptosis. Importantly, SEL24-B489 was not toxic for cells derived from healthy donors.
Since microenvironmental cues increase expression of PIM kinases, we hypothesized that interactions with stromal cells might hinder the in vitro activity of the PIM inhibitor. To explore this possibility, we compared apoptotic response to SEL24-B489 in CLL cells co-cultured on HS5 monolayers and CLL cells grown without the stromal support. In 6 out of 7 tested cases, SEL24-B489 overrode the protective signals from HS5 cells and induced apoptosis, although the cytotoxic effect of PIM inhibitor was stronger in the absence of stromal cells.
PIM1 was shown to regulate CLL cells migration through CXCR4(S339) phosphorylation (Mol Cancer Ther 2014, 13: 1231-45). Accordingly, SEL24-B489 decreased phospho-CXCR4(S339), CXCR4 surface expression, and impaired CLL cells migration in the CXCL12 gradient. Surprisingly, decrease in the CXCR4 surface expression after SEL24-B489 was relatively modest when compared to the effect of this inhibitor on CXCL12-directed migration. We found that incubation of CLL cells with CXCL12 led to increase in the phosphorylation of mTOR(S2448) and Akt(S473). SEL24-B489 reduced the levels of p-mTOR(S2448), p-Akt(S473), p-4EBP1(T37/T46) and p-TSC2(S1798), revealing inhibitory effect on mTOR pathway. Pre-incubation of CLL cells with an mTOR inhibitor similarly restrained CXCL12-mediated mTOR activity and led to impaired CLL cells migration, uncovering the key role of mTOR axis in CXCR4-dependent migration. Thus, SEL24-B489 impairs the CLL cell migration by inhibiting CXCR4 surface expression and the CXCR4-triggered mTOR pathway.
Taken together, we show that microenvironment signals increase expression of PIM kinases, supporting CLL cell survival and migration. Inhibition of PIM kinases impairs CXCR4-dependent migration and leads to CLL cells death, regardless of the p53 status. Targeting PIM kinases in CLL patients will likely release the cells from microenvironmental niches and might be a rational therapeutic strategy.

Disclosures
Warzocha: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Czardybon:Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Consultancy, Membership on an entity's Board of Directors or advisory committees.