Pharmacological IGF1R/IRS Inhibitor, NT157, Effectively Induces Apoptosis and CDKN1A Expression in Acute Lymphoblastic Leukemia Cells
Authors: Rodrigues Alves Ana Paula Nunes , Machado-Neto João Agostinho , Fernandes Jaqueline Cristina , Fenerich Bruna Alves , da Silva Fernanda Borges , Scheucher Priscila Santos , Simões Belinda Pinto , Rego Eduardo Magalhães , Traina Fabiola 
Published: October 2019

Abstract
Background: Acute lymphoblastic leukemia (ALL) is an aggressive cancer of immature progenitors that shows aberrant activation of signaling pathways. The IGF1/IGF1R signaling pathway is initiated through binding of the ligand (IGF1) to its transmembrane receptor (IGF1R), and subsequent activation of IRS1 and IRS2 proteins activating PI3K/Akt/mTOR and MAPK pathways, which are important signaling pathways reported to contributes to pathogenesis of ALL. However, the therapeutic potential of IGF1R/IRS signaling has not been previously studied in ALL cells.
Aims: We herein aimed to investigate the effects of the pharmacological IGF1R/IR and IRS1/2 inhibition in ALL cells.
Materials and Methods: T-ALL Jurkat and MOLT4, and B-ALL Namalwa and Raji cell lines were used. Peripheral blood or bone marrow mononuclear cells from ALL patients (T-ALL [n=2] and B-ALL [n=2]) at the time of diagnosis or relapse were used for functional studies and compared with health donors (n=2). Cell lines were treated or not with the IRS1/2 pharmacological inhibitor NT157, at 0.2, 0.4, 0.8, 1.6 and 3.2 µM, or with the IGF1R/IR inhibitor OSI-906, at 1, 5, 10 and 20 µM for 24, 48 and 72 hours. After drug exposure, cell lines were evaluated for cell viability (MTT assay), apoptosis (annexin V/PI and cleavage caspase 3), clonogenicity (colony forming assay), and protein expression/activation (Western blot) and PCR-array for MAPK signaling. Primary ALL cells were culture with IL7 (100 ng/mL), IL3 (30 ng/mL), SCF (30 ng/mL) and FLT3L (100 ng/mL) in the presence or not of NT157 or OSI-906 for 72 hours and them submitted to cell viability and apoptosis assays. Statistical analyzes were performed by the ANOVA or Student t test. P value <0.05 was considered statistically significant.
Results: In ALL cell lines, NT157 treatment above 0.4 µM at 48 hours and 72 hours decreased cell viability in a dose and time-dependent manner (all p<0.05). Using a nonlinear regression analysis, IC50 cytotoxicity for Jurkat, MOLT4, Namalwa and Raji at 72 hours was 0.3, 0.9, 1.8, and 1.9 µM, respectively. NT157 significantly induced apoptosis in a dose and time-dependent manner (all p<0.05). The highest percentage of apoptotic cells were observed upon NT157 1.6 µM at 72 hours for all cell lines (Control vs. NT157 1.6 µM: 6% vs. 87% for Jurkat, 16% vs. 95% for MOLT4, 4% vs. 41% for Namalwa, and 25% vs. 50% for Raji cells). Western blot analysis revealed increased cleaved-caspase 3 levels in all cell lines. Jurkat and Raji cells were tested for clonogenicity; colonies were counted after 8 days. The number of colonies reduced by 14%, 14% and 23% for Jurkat and by 32%, 44% and 77% for Raji cells at the dose of 0.4, 0.8, and 1.6 µM, respectively. Western blot analysis revealed that NT157 treatment induced IRS1 down-regulation in a dose-dependent manner after 24 hours of treatment. In Jurkat cells, PCR-array analysis reveals that NT157 modulates 25 genes, including downregulation of MYC (proliferation-related genes) and upregulation of CDKN1A, CDKN1C and CDKN2A (cell cycle arrest-related genes), and JUN and FOS (apoptosis-related genes), validation experiments confirmed an upregulation of CDKN1A, JUN and FOS in all ALL cell lines upon NT157 treatment. Notable, IRS1/2 pharmacological inhibition by NT157 reduced cell viability of primary ALL cells and was a potent apoptosis inductor. Upon NT157 1.6 µM, cell viability for T-ALL and B-ALL primary cells was inhibited by 55±5% and 15±8%, respectively. The apoptosis rates for untreated cells and 1.6 µM NT157 was 12±8% and 59±27% for T-ALL primary cells and 19±2% and 37±1% and B-ALL primary cells, respectively. NT157 treatment did not presented citotoxicity in peripheral blood mononuclear cells (PBMC) from healthy donors. On the other hand, in ALL cell lines tested, OSI-906 treatment reduced cell viability, but did not induce apoptosis; higher doses of OSI-906 were necessary to induce cytotoxicity. OSI-906 did not modulate viability and apoptosis of primary ALL cells and normal PBMC.
Conclusion: Our results revealed that pharmacological inhibition of IRS1/2, by NT157, exerts a cytotoxic effect in ALL cell lines and primary ALL cells, while IGF1R/IR inhibition, by OSI -906, has a predominant cytostatic effect only at higher doses. These data indicate that direct inhibition of IRS proteins by NT157 induces better antileukemic effects compared to OSI -906, and targeting IRS proteins may be an effective anti-ALL approach.

Disclosures
No relevant conflicts of interest to declare.