Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal B lymphocytes. For decades, nucleoside analogs, alkylating agents, and immunotherapeutics have remained the mainstay in treating this disease. Despite major advances in this field, CLL remains incurable with standard therapy. In recent years, preclinical and early clinical data on the use of kinase inhibitors have sparked new hope in the treatment of CLL. The multikinase inhibitor sorafenib, targeting RAF, platelet-derived growth factor receptor (PDGFR), KIT, FMS-like tyrosine kinase 3 (FLT3), and vascular endothelial growth factor receptor (VEGFR), has been approved for the treatment of renal cell carcinoma and hepatocellular carcinoma. Recent studies suggested that CLL cells might be also susceptible to this compound, however the precise mode of action in CLL cells remains elusive. In this study, we identified the Insulin-like growth factor receptor-I (IGF1R) pathway as novel target of sorafenib inducing cell death in CLL cells.
Treatment with 10 μM of sorafenib significantly increased apoptosis in primary CLL cells as determined by AnnexinV/PI staining via flow cytometry. Commensurate with its RAF inhibiting properties, the apoptotic effect of sorafenib was accompanied with ERK pathway inhibition. Moreover, sorafenib treatment decreased phosphorylation of SRC and AKT, molecules implicated with IGF1R and insulin receptor (IR) signaling. Interestingly, the latter were strongly expressed in primary CLL cells compared with healthy B cells. Similar to sorafenib, 24 hour treatment of CLL cells with the three structurally distinct IGF1R inhibitors Picropodophyllin, AG1024, and Linsitinib significantly increased apoptosis compared with vehicle control resulting in decreased phophorylation of MEK, ERK, SRC, and AKT. Sorafenib and the IGF1R inhibitor AG1024 also downregulated the expression of IGF1R on CLL cells but not on healthy B cells.
To test whether sorafenib modulates IGF-1 binding and thereby influences the IGF1R activation, we biotinylated recombinant IGF-1 and tested its binding to the IGF1R via flow cytometry. We observed a reduced IGF-1 binding after sorafenib treatment. IGF-1 binding after treatment with different IGF1R inhibitors was performed as an internal control. In order to further establish the functional relevance of IGF1R expression in CLL, we performed IGF1R specific and non-silencing siRNA experiments in primary CLL cells. In line with our previous results, IGF1R knockdown resulted in a significant decrease of cell viability and in downregulation of RAF-1 expression, and MEK, ERK, SRC, and AKT phosphorylation. The stromal microenvironment protects CLL cells from spontaneous and drug-induced apoptosis. Sorafenib, AG1024, and Picropodophyllin counteracted the protective effect of microenvironmental factors simulated by the presence of the murine stromal cell line M210B4, the chemokine CXCL12, and the integrin CD49d.
Finally, we used the Eμ-Tcl1 transgenic mouse model to validate these results in vivo. Male and female mice (n=8) were treated with 25 mg/kg of the IGF1R inhibitor Linsitinib per oral gavage for 7 days and the amount of CD5/CD19 positive cells was determined flow cytometrically at different time points. We observed a reduction of CD5/CD19-positive cells by 26,1% and 23,2% after 4 and 8 days of treatment, respectively.
Our results provide a novel mechanism of action of the multikinase inhibitor sofarenib in CLL cells by blocking IGF1R mediated signaling. IGF1R inhibition by itself induced apoptosis in CLL cells in vitro and in vivo, thus identifying IGF1R as promising target for therapeutic approaches and proposing IGF1R inhibitors for clinical assessment in the therapy of CLL.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.