Abstract
The classical Hodgkin lymphoma (cHL) is characterized by a presence of rare malignant Reed-Sternberg (RS) cells surrounded by abundant reactive infiltrate. Tumor RS cells express multiple cytokines, chemokines and immunoregulatory proteins, such as PD-L1 or galectin-1, that enhance recruitment of the infiltrating cells and allow malignant cells to escape from host immune surveillance. Since targeting these immunomodulatory molecules is a highly promising therapeutic strategy in cHL, identification of pathways and mechanisms orchestrating tumor immune evasion and supporting RS cell survival can further reveal targetable vulnerabilities of cHL. Expression of immunoregulatory proteins in cHL is modulated by tumor-specific activity of certain pro-survival transcription factors, such as NFκB and STATs. Since the activity of these transcription factors is modulated by oncogenic PIM1/2/3 serine/threonine kinases, we hypothesized that PIMs may support RS cell survival and foster their immune privilege. For these reasons, we investigated PIM1/2/3 expression in cHL and determined mechanisms underlying their expression. Furthermore, we assessed impact of PIM inhibition on expression of key factors engaged in development of the immunosuppressive microenvironment and HRS cell survival.
Our analyses revealed that PIM1/2/3 are ubiquitously expressed in primary and cultured RS cell lines. At least one PIM isoform was expressed in each cell line and in 97% of 67 primary cHL biopsies. RS cells treated with JAK1/2/3 inhibitor exhibited reduced PIM1 and PIM2 levels. Genetic inhibition of canonical NFkB activity with IkB super-repressor or shRNA-mediated ablation of alternative NFkB pathway led to decrease of PIM2 and PIM3, suggesting that PIM-1/2/3 expresion in cHL depends at least in part on JAK-STAT and NFkB activity.
To assess the role of PIM kinases in cell viability, we silenced expression of each PIM isoform (individually or simultaneously) in HDLM-2 cells. Knockdowns of individual PIM isoforms were associated with marked increased in remaining isoforms expression and were not associated with toxicity. In marked contrast, downregulation of all three isoforms increased cellular apoptosis by 17%. For this reason, for subsequent studies we used a newly developed pan-PIM inhibitor (SEL24-B489). The inhibitor was toxic to all cells with IC50 ranging from 3-5 µM. To determine mechanisms underlying toxicity, we assessed the activities of specific PIM substrates: 4EBP1, S6, and p65 (RelA). SEL24-B489 rapidly decreased PIM-dependent phosphorylation of these molecules in all tested cell lines. Furthermore, it reduced DNA binding activity of the NFκB-p65 complexes, indicating that PIM kinases modulate NFκB activity in cHL. For this reason, we next assessed the consequences of PIM inhibition on NFκB-dependent transcription. SEL24-B489 significantly downregulated mRNA of NFkB target genes associated with HRS cell survival and proliferation, such as Bfl-1, RelB and CD40. In cells treated with PIM inhibitor SEL24-B489 we also found markedly decreased expression NFkB-dependent cytokines and chemokines specifically shaping pro-tumoral microenvironment, such as IL-8, CCL5 and IL-13. In addition, cell lines exposed to SEL24-B489 treatment exhibited decreased expression of immunomodulatory PD-L1 and Gal-1 proteins. Finally, we investigated the efficacy of SEL24-B489 in vivo in the murine xenograft model using HDLM-2 cells. In contrast to animals exposed to vehicle alone, we observed inhibition of tumor growth by 95,8% in SEL24-B489-treated animals (p=0,0002). Consistent with the in vitro data, we observed strong downregulation of phospho-S6, GAL-1 and PD-L1 proteins in tumor sections from PIM inhibitor-treated animals.
Taken together, we demonstrated that the oncogenic PIM-1/2/3 kinases are expressed in RS cells and their activity can be specifically blocked using a pan-PIM inhibitor SEL24-B489. PIM inhibition significantly reduced activity of specific PIM substrates and decreased the expression of NFκB-dependent pro-survival genes and key immunomodulatory proteins. These results highlight the pleiotropic activity of SEL24-B489 and indicate that PIM kinases are promising therapeutic targets in cHL.
Disclosures
Czardybon: Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment.
Dissecting the binding mechanisms of transcription factors to DNA using a statistical thermodynamics framework