IGF Signaling Predicts Outcomes and Is a Promising Target Therapy for Acute Myeloid Leukemia

Background : Despite the growing knowledge about acute myeloid leukemia (AML) pathophysiology, it remains a disease with high mortality rates, and the early recognition of patients at higher risk allows adjusted risk- or target-specific therapies. In AML, autocrine insulin like growth factor type 1 (IGF1) and its receptor (IGF1R) activation has been previously described. Free IGF mediate cellular functions through binding to the two IGF receptors, IGF1R and IGF2R. IGF2R binds to and induces degradation of IGF2, preventing IGF2-IGF1R signaling activation, IGF1R activation induces cell proliferation and prevents cell death. Therefore, IGF2R may act as a tumor suppressor and IGF1R has opposite effects. IGF1R pharmacological inhibitors have recently been investigated in the clinical setting for solid tumors, but still underexplored in hematological neoplasms. Aims : To investigate IGF1R and IGF2R mRNA expression in primary samples from AML patients and their impact in clinical outcomes, and to analyze the effects of the IGF1R pharmacological inhibitor linsitinib in AML cell lines. Material and methods : IGF1R and IGF2R mRNA expression levels in AML patients (n=172) were obtained from TCGA AML study (2013) available online on CBioPortal for Cancer Genomics (Cerami et al. 2012; Gao et al. 2013). Gene expression was investigated, by qPCR, in a subset AML cohort (n=101; APL=48, CBF-leukemia=53 [t(8;21)=30, Inv16=23]), and in total bone marrow samples from healthy donors (HD) (n=23). The study was approved by the ethics committee of the Institution. NB4, NB4-R2 and Kasumi-1 cell lines were submitted to linsitinib (0.25, 0.5, 1.0, 2.5, 5.0, 10, 20 or 40 µM) for 72 hours and were evaluated for cell viability (MTT assay), apoptosis (Annexin V/PI), autophagy (acridine orange staining), and protein expression/activation (western blot). Statistical analyses were performed using Student's t-test, Mann-Whitney test, Kruskal-wallis test and Spearman correlation test, as appropriate. For survival analysis, Kaplan-Meyer curves were compared with log-rank test and cox regression analyzes were applied. Results :Patients were dichotomized according to the median IGF1R and IGF2R expression values, and the IGF1R / IGF2R ratio was calculated by division of absolute number of reads coming from RNA-seq data. Patients with high IGF1R / IGF2R showed poorer overall survival (OS) (29% vs. 56%, P <0.01), and disease free survival (DFS) (31% vs. 45%, P =0.015). Individually, high IGF1R (33% vs. 51%; P =0.042) and low IGF2R (32% vs. 52%; P =0.012) were associated with a shorter OS. By Cox regression analysis, IGF1R / IGF2R ( P= 0.039), cytogenetic risk stratification ( P <0.001), age ( P <0.001) and white blood cell counts ( P =0.003) independently predicted poorer OS. AML patients harboring t(15;17) or t(8;21) presented higher levels of IGF1R / IGF2R when compared with other AML patients ( P≤ 0.01). Using a subset of APL and CBF-leukemia, a higher IGF1R / IGF2R ratio was observed in AML patients with t(15;17) or t(8;21) compared with healthy donors (HD) ( P <0.01). IGF1R and IGF2R expression positively correlated in HD ( r =0.9296; P <0.001), and negatively correlated in AML TCGA study ( r =-0.2366; P =0.0018). In NB4, NB4-R2 and Kasumi-1 cells, linsitinib ≥1 µM reduced cell viability ( P <0.05) and significantly increased apoptosis, as demonstrated by increased cleaved caspase 3 and annexin V+ cells. The mean percentage of annexin V+ cells for control, linsitinib 10, 20 and 40 µM were 3,9%, 14%, 32% and 93% for NB4, 6%, 24%, 53% and 96% for NB4-R2 and 7%, 19%, 49% and 96% for Kasumi-1 (all P <0.05), respectively. Linsitinib 10 µM increased autophagy in at least 3 fold-change in all cell lines tested (all P <0.05). Linsitinib 10 µM plus the autophagy inhibitor chloroquine 20 µM increased apoptosis when compared with monotherapy in NB4 cells (45% vs 15%), NB4-R2 cells (68% vs 15%) and Kasumi-1 (35% vs 15%) (all P <0.05). The IGF1R pharmacological inhibitor reduced phosphorylation/activation of IGF1R (Tyr1135), IRS1/2 (Tyr612), AKT1/2/3 (Ser473), P70S6K (Thr421/Ser424), 4EBP1 (Thr70) and ERK1/2 (Thr185/Tyr187). Conclusions :In AML,increased IGF1R / IGF2R predicts poorer survival outcomes and the pharmacological IGF1R inhibitor linsitinib exerts an anticancer activity and downregulates PI3K/AKT and MAPK signaling. In the concept of multi target treatment, IGF1R arise as a promising therapeutic target for patients with AML.