The Effect of Insulin-Like Growth Factor 1 on Waldenstrom Macroglobulinemia
Authors: Melhem Molly R. , Roccaro Aldo M. , Farag Mena , Azab Abdel kareem , Ngo Hai T. , Sacco Antonio , Jia Xiaoying , Azab Feda , Runnels Judith , Burwick Nicolas , Quang Phong , Husu Emanuel N. , Teru Hideshima , Anderson Kenneth C. , Leleu Xavier , Ghobrial Irene M. 
Published: April 2020

Abstract
Background: Waldenstrom’s Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Insulin-like growth factor 1 (IGF1) is a polypeptide hormone that has been shown to have a proactive role in many cancer cell types, including multiple myeloma and solid tumor cells. We evaluated the role of IGF1 in WM.
Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Bone marrow-derived primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. The small molecule IGF-1R inhibitor II (Calbiochem) and the inhibitory antibody αIR3 (Calbiochem) were used. Cytotoxicity and DNA synthesis were measured by MTT assay and thymidine uptake assay, respectively. Cell signaling and apoptotic pathways were determined by Western Blot. Cell cycle and receptor analysis was obtained through flow cytometry. IGF1 levels were measured by ELISA. Receptor tyrosine kinase activity was evaluated in WM cell lines using the Luminex-microbeads-based assay.
Results: We demonstrated that the IGF1 receptor (IGF1R) was expressed on WM primary patients and normal CD19+ control B cells, while IGF1 levels of serum and bone marrow samples in both patient and healthy donor samples were similar, suggesting a possible constitutive activation of the pathway downstream of IGF1R in WM and independent of receptor activity or cytokine levels. Surface IGF1R was expressed on BCWM1 cells, in serum-starved conditions, while, it was internalized with the addition of FBS that includes IGF-1. Finally, we also showed that IGF1 induces the activation of IGF1R as demonstrated by the induction of related tyrosine kinase activity. To demonstrate the protective effect of IGF1 on WM cells, IGF1 was added to serum starved BCWM1 cells, and we found it rescued nearly 100% of the cells from apoptosis in concentrations as low as 25ng/ml. The effect of IGF1R inhibitor on WM cells has been investigated and found that it blocked migration, induced cytotoxicity and decreased cell proliferation; without any effect on healthy donor peripheral blood mononuclear cells. Furthermore, the inhibitor decreased the tyrosine kinase activity of IGF1R, overcoming its initial activation in the presence of IGF1.
Conclusion: IGF1 plays a complex role in WM cells with no evidence of constitutive activation of the receptor itself or higher levels of cytokine in WM marrow samples, indicating that activation of this pathway is downstream of IGF1R. Inhibition of IGF1R by a specific small molecule or antibody inhibitor led to a significant decrease in proliferation, survival, and migration in WM cells, but not in control mononuclear cells. These studies provide the framework to investigate the role of IGF1R inhibitors in clinical trials in WM.