Background : Mutations of the FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) occur in approximately in 30% of AML patients, and is considered a driver mutation that presents a high leukemic burden and confers a poor prognosis. Insulin-like growth factor 1 receptor/insulin receptor substrates (IGF1R/IRS) signaling has been implicated to the malignant phenotype in myeloid neoplasms. However, how extend is the participation of IGF1R/IRS signaling in FLT3-mutated transformation is still an open question. Aims: To determine the clinical impact of IGF1R-IRS1/2 signaling component in FLT3-mutated AML patients and to investigate the therapeutic opportunity of pharmacological inhibition of IGF1R-IRS1/2 in preclinical models of FLT3-mutated AML. Material and methods: Clinical and molecular data from AML patients were obtained from Gene Expression Omnibus (GEO-GSE 6891; n=406, median age 44 years) and TCGA AML study (n=173, median age 58 years). For survival analysis in FLT3-mutated patients, IGF1R, IGF2R, IRS1 and IRS2 mRNA expression levels from 150 and 49 patients from GSE6891 and TCGA patients were included, respectively. MOLM-13 and MV4;11 FLT3-mutated cell lines were submitted to FLT3 inhibitors (midostaurin and quizartinib) and/or Linsitinib (IGF1R/IR inhibitor: 0.5 to 40 µM) or NT157 (IGF1R-IRS1/2 inhibitor: 0.25 to 10 µM) until 72 hours and evaluated for cell viability (MTT), apoptosis (Annexin V/PI), proliferation (CFSE), and protein expression/activation (western blot and proteomics). Statistical analyses were performed using ANOVA or Mann-Whitney. For survival analysis, Kaplan-Meier curves were compared with the log-rank test, and multivariate analysis was performed by Proportional Hazard Cox regression. Results: IGF1R, INSR, IGF1, IGF2, IRS1, and IRS2 were all downregulated in FLT3-mutated in comparison with wild-type in both AML cohorts (all P<0.05). Higher IGF1R expression (overall survival [OS] median time [MT]: low 23.6 months [mos] vs. high 8.9 mos, P=0.003; disease-free survival [DFS]: 34.2 vs. 10.5, P=0.03), as well as reduced IRS2 (OS: 8.3 vs. 17.2, P=0.001; DFS: 8.8 vs. 28.2, P=0.05) expression were associated with shorter disease-free survival and overall survival in GSE 6891 cohort. In the TCGA cohort, higher IGF1R expression predicted shorter DFS (20.6 vs. 7.3, P=0.03) but did not predict OS. Both linsitinib and NT157 significantly reduced cell viability in MOLM13 (IC50 for Linsinitinb 24, 48 and 72 hours:12.6, 11.2 and 8.1 µM, and for NT157: 0.9, 0.8 and 0.7 µM) and MV4;11 cells (IC50 for Linsinitinb 24, 48 and 72 hours:14.9, 11.2 and 9.5 µM, and for NT157: 1.2, 1.4 and 1.0 µM). Linsitinib (>5µM) and NT157 (>0.5 µM) significantly induced apoptosis in a dose-dependent manner in both cell lines (all P<0.05), but showed no additive effect in cotreatment with midostaurin and quizartinib, suggesting that both classes of inhibitors acting in similar intracellular targets. As demonstrated by CFSE assay, linsitinib (>2.5 µM) and NT157 (>0.5 µM) significantly reduced cell proliferation in a dose-dependent-manner. Linsitinib and NT157 inhibited IGF1R and IRS1/2 activation, as well as its related signaling pathways as demonstrated by reduced activation/expression of FLT3 (Y591), AKT1/2/3 (S473), mTOR (S2448), STAT5 (Y694). Linsitinb acted as a cytostatic drug by inducing autophagy, as determined by mTORC1 inhibition and mTORC2 activation, as well as p62 degradation and LC3B-I conversion into LC3BII. NT157 was more cytotoxic activating AP-1 system, as evidenced by p-JNK/SAPK and P38 MAPK and more prominent cleavage of CASP3 and PARP1 and reducing BCL-xL expression. A better characterization of the molecular context of IGF1R-IRS1/2 pharmacological inhibition will be allowed by proteomic of total extract of MOLM13 and MV4;11 cells treated with linsitinb (10 µM) or NT157 (1 µM) that will be presented at the meeting. Midostaurin ≥2.5 nM and quizartinib ≥0.5 nM treatment abolished IGF1R activation and IRS1 and IRS2 expression, as well as induced autophagy as demonstrated by the degradation of p62 and accumulation of Beclin 1. Conclusion: In FLT3-mutated AML, expression of IGF1R-IRS1/2 signaling components are deregulated and high IGF1R expression predicted dismal prognosis. Pharmacological inhibition of IGF1R-IRS1/2 exerted antineoplastic activity in cellular models and arises as a promising therapeutic target for FLT3-mutated AML patients.
Disclosures
No relevant conflicts of interest to declare.
Prognostic Value of the Preoperative Lymphocyte-to-monocyte Ratio for Survival After Lung Cancer Surgery
