The Pharmacological IGF1R-IRS1/2 Inhibitor NT157 Presents Multiple Anti-Neoplastic Effects in Myeloproliferative Neoplasms

Introduction: The recurrent V617F mutation in JAK2 plays an important role in the pathogenesis of BCR-ABL1 negative myeloproliferative neoplasms (MPN). Absence of complete clinical response to ruxolitinib, a JAK1/2 inhibitor, highlights the need to target other signaling pathways that contribute to JAK2. Recent evidence indicates that the IGF1R/IRS pathway is a potential target in MPN: (i) insulin receptor substrate 2 (IRS2) cooperates with malignant transformation induced by JAK2^V617F, and (ii) increased IGF1 signaling induced by AIRAPL down-regulation causes MPN in mice. NT157 is a synthetic compound designed as an IRS1/2 inhibitor, which presented anti-neoplastic effects in solid tumors. Aims: The objective herein was tocharacterize the molecular and cellular effects of NT157 treatment, combined or not with ruxolitinib, in MPN JAK2^V617F. Methods: HEL and SET2 cell lines (both carrying JAK2^V617F mutation) were treated or not with vehicle, NT157 and/or ruxolitinib and submitted to evaluation of cell viability (MTT assay), apoptosis (annexin V/PI and caspase cleavage), proliferation (Ki-67), clonogenicity (colony forming assay), cell cycle (PI), gene expression (PCR array and qPCR) and protein expression/activation (Western blot). Primary cells from polycythemia vera (PV) patients (n=3) were exposed to NT157 (3.2 µM) treatment and evaluated for erythropoietin-independent colony formation. Statistical analysis was performed using ANOVA or Student's t test. P -value <0.05 was considered statistically significant. Results: NT157 treatment significantly decreased cell viability, clonogenicity and cell proliferation (all p <0.05). NT157 induced apoptosis at doses equal or higher than 0.8 μM as verified by flow cytometry analysis of annexin V/PI ( p ˂0.001) and confirmed by increased levels of cleaved caspase 3, 8 and 9. The percentage of apoptotic cells (annexin V positive) identified for untreated cells and cells exposed to NT157 0.8 μM was respectively, 14 and 56% after 24 hours of treatment; 11 and 60% after 48 hours, 6 and 40% after 72 hours (all p ˂0.001). Cell cycle analysis was performed 24 hours after NT157 exposition and revealed cell cycle arrest in G₂/M, associated with decrease in G₀/G₁ population (p ˂0.001). In a molecular scenario, NT157 treatment strongly reduced IRS1 and IRS2 expression, and STAT3, STAT5 and ERK1/2 phosphorylation in a dose dependent manner. PCR array analyzes revealed that NT157 treatment modulated 23 oncogenes and tumor suppressor genes. Based on the cellular effects observed after NT157 treatment, we selected six genes, three repressed oncogenes and three upregulated apoptotic-related genes, to be validated in a larger number of experiments. Upon NT157 exposition, CCND1 (cyclin D1), MYB and WT1 mRNA levels were reduced, while CDKN1A (p21), FOS and JUN mRNA levels increased (all p ˂0.05). The combined treatment of NT157 plus ruxolitinib did not present potentializing effects when compared to the monotherapies. In contrast, the combined treatment prevented, at least in part, the cytotoxic effects of NT157. In HEL cells, NT157 0.8 µM monotherapy promoted enhanced apoptosis rate compared to the combined treatment, using NT157 0.8 plus ruxolitinib 0.3 µM (52% vs. 38%, respectively [ p ˂0.05]). Ruxolitinib also prevented NT157 effect on cell cycle progression, causing a reduction in G₂/M arrest when compared to NT157 monotherapy (p <0.05). In primary hematopoietic cells from PV patients, NT157 exposition reduced erythropoietin-independent colony formation by 49 % (patient #1), 12% (patient #2) and 23% (patient #3); the mean reduction of erythroid colony number for all PV samples was 29% (p <0.05). Conclusions: IGF1R-IRS1/2 pharmacological inhibitor NT157 displayed remarkable anti-neoplastic effects in JAK2^V617F cells lines and MPN primary cells. Our exploratory approaches provided new insights about molecular mechanisms of NT157. Our findings indicate that inhibition of IGF1R/IRS signaling, using NT157 monotherapy, emerges as a novel therapeutic opportunity for MPN JAK2^V617F.