Lumiliximab Triggers Apoptosis Mechanisms in CLL Cells through the Inhibition of PI3-K/Akt Pathway.
Authors: Shehata Medhat , Schnabl Susanne , Ponath Elena , Tauber Stefanie , Demirtas Dita , Hilgarth Martin , Bilban Martin , Badrnya Sigrun , Fleiss Karin , Schwarzmeier Josef D. , Gaiger Alexander , Hubmann Rainer , Zielinski Christoph C , Hughes Steve , Maclaren Ann , Jaeger Ulrich 
Published: October 2019

Abstract
Abstract 2371
Poster Board II-348
Chronic lymphocytic leukemia (CLL) is a clonal expansion of B cells which is characterized by a defect in apoptosis and is associated with the co-expression of CD19, CD5 and CD23. Lumiliximab is a monoclonal antibody against CD23 which has been shown to exert a promising therapeutic effect in CLL in vivo and to induce apoptosis in CD23+ lymphoma cells in vitro. The aim of this study was to investigate the direct effect of lumiliximab on cell viability and to explore its molecular mechanism of action in primary CLL cells. PBMC from twenty CLL patients were used in this study. Nine patients had previous therapy and 11 were untreated. Nine patients had unmutated and 11 had mutated IgVH genes. The mean percentage of CD19+/CD5+/CD23+ cells was 80% (range 53-98%). PBMC were exposed to various concentrations of lumiliximab (1-50 μg/mL) for various durations (1-15 days). The long term incubation with lumiliximab was performed in a microenvironment co-culture model using primary human stromal cells which prevent spontaneous apoptosis of CLL cells. Cell viability was assessed by flow cytometric analysis using annexin V/PI staining and by MTT assays. The results showed that single exposure to lumiliximab had a minimal effect on cell viability (< 1 fold increase in apoptosis rate compared to untreated cells and to the isotype control antibody). Cross-linking with goat anti-human IgG was more effective in inducing cell death. Interestingly, repeated exposure to lumiliximab without cross-linking had a significant pro-apoptotic effect selectively in the CD19+/CD5+ cells. Western blotting analysis demonstrated a significant biological response to the single and repeated exposure to lumiliximab in spite of the moderate pro-apoptotic effect. Lumiliximab induced a significant decrease in Bcl-2, Mcl-1 and Hsp70 protein expression. In addition, it resulted in a significant decrease in the phosphorylation of Akt1 at serine residue 473 and dephosphorylation (activation) of the tumor suppressor PTEN at serine residue 380 suggesting the involvement of the PI3-K/Akt/PTEN cascade in priming CLL cells to undergo apoptosis by lumiliximab. Pre-incubation of CLL cells with lumiliximab enhanced the pro-apoptotic effect of PI3-K inhibitor LY292004 and the CK2 inhibitor apigenin. Consistent with previous observations, pre-exposure of CLL cells to lumiliximab augmented the cytotoxic effect of Fludarabine. The in vitro response to lumiliximab did not appear to be influenced by IgVH mutation status, cytogenetics or by previous therapy. To gain further insight into the downstream targets of lumiliximab in CLL, microarray analysis and pathway exploration was performed. The data revealed that lumiliximab regulates several sets of genes which are involved in chemokine signaling, cytokine/cytokine receptor interaction, oxidative stress, PI3-K and integrin signaling, complement cascade and Toll-like receptor signaling. Single exposure to lumiliximab led to down-regulation of several genes including LY9, GPR183, RGS1, HSPA6, and INPP5F and up-regulation of FN1, FAIM3 and LOX. Repeated exposure to Lumiliximab led to a significant down-regulation of TXNIP, CTSB, SODS, IFI44, IRF7, TNFSF13B, MS4A7, CCL4, CCL7, CCL8, cathepsin B, MARKS, ADAMS and FCER1G and up-regulation of SPP1, C10orf10, ANGPTL6, RBBP4 and GSTA4. In conclusion, these data demonstrate that repeated exposure to Lumiliximab is effective in priming CLL cells to undergo apoptosis through inactivation of the PI3-K/Akt pathway and render the cells more sensitive to cytotoxic compounds. The data also provide further evidence of a promising therapeutic role for lumiliximab in CLL and a rationale for lumiliximab-based drug combinations to improve treatment of this incurable disease.

Disclosures:
Shehata: Biogen Idec: Research Funding. Hughes:Biogen Idec: Employment. Maclaren:Biogen Idec: Employment. Jaeger:Biogen Idec: Research Funding.