Pharmacological IRS1/2 Inhibition Reduces Cell Viability in BCR-ABL1 Positive Cells

Introduction: Chronic myeloid leukemia (CML) is a hematological malignancy associated with the BCR-ABL1 fusion gene, which drives the proliferative disease phenotype by activating multiple signaling pathways. Most CML cases are successfully treated with tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1. However, in some cases, drug resistance limits TKIs efficacy, and the identification of other crucial proteins in the BCR-ABL1 signaling pathways may contribute to optimize anti-CML approaches. IRS1 mRNA expression has been previously identified as positively correlated with overall survival in BCR-ABL1-positive adult acute lymphoblastic leukemia. In K562 cells, IRS1 has been identified as a binding partner of BCR-ABL1 protein and was capable of activating PI3K/Akt/mTOR and MAPK pathways. Recently, a pharmacological IRS1/2 inhibitor (NT157) has been developed and has shown promising results in preclinical studies on solid tumors. We herein aimed to investigate IRS1 and IRS2 expression and the effects of IRS1/2 inhibition on cell proliferation, apoptosis and clonogenicity in BCR-ABL1 positive and normal hematopoietic cells.

Materials and Methods: Total bone marrow cells from healthy donors (n=11) and CML patients at the time of diagnosis (n=24) were submitted to gene expression analysis by quantitative PCR with specific primers for IRS1, IRS2 and β-actin. All subjects provided informed written consent and the study was approved by the ethics committee of the Institution. K562 cells were submitted to IRS1/2 pharmacological inhibition using NT157 (0.2, 0.4, 0.8, 1.6, 3.2 and/or 6.4 µM) for 24, 48 and 72 hours and were evaluated for cell viability (MTT assay), proliferation (Ki-67), apoptosis (Annexin V/PI), and protein expression/activation (Western blot). Alternatively, cells were submitted to IRS1 and IRS2 gene silencing using specific shRNA lentiviral delivery, and submitted to functional studies. NT157 effects were analyzed by in vitro hematopoietic colony formation of bone marrow cells from two patients with CML at diagnosis, and of normal cord blood cells from one individual. Cells were seeded at 4.5x10⁴ per well in a culture system for 14 days. Statistical analyses were performed by Student's t-test or Mann-Whitney test, as appropriate.

Results: IRS1 and IRS2 mRNA expression was similar between normal donors and CML samples (p ≥.05). NT157 treatment reduced K562 cell viability in a time and dose-dependent manner; using a nonlinear regression analysis, IC50 for cytotoxicity was 9.8, 0.6 and 0.68 µM for 24, 48 and 72 hours, respectively. NT157 0.8 and 3.2 µM reduced cell viability in 14% and 19% at 24 hours, 50% and 61% at 48 hours and in 59% and 68% at 72 hours of treatment (all p <.05). After 48 hours of NT157 exposure, Ki-67 staining revealed a decrease in cell proliferation by 10% at 0.8 µM, 40% at 1.6 µM, and 75% at 3.2 µM. Pharmacological IRS1/2 inhibition significantly induced apoptosis as noted by increased cleaved caspase 3 and 9 by Western blotting analysis, and AnnexinV/PI staining. The percentage of apoptotic (AnnexinV+) cells for control, NT157 0.8 and 3.2 µM at 48 hours of treatment were 15%, 38% and 61%, respectively (p<.05). Upon NT157 0.8, 3.2 and 6.4 µM, colony formation of CML primary cells was inhibited by 7%, 36% and 78% for patient #1, and by 29%, 19% and 62% for patient #2, with a reduction predominance in granulomonocytic colonies for both patients. Interestingly, NT157 treatment did not inhibit colony formation of committed normal cord blood cells; the number of colonies for control, NT157 0.8, 3.2 and 6.4 µM were 88, 84, 97, and 92, respectively. Of note, lentiviral-mediated silencing of IRS1, but not of IRS2, significantly decreased K562 cell viability.

Conclusion: Although IRS gene expression analysis did not differ between CML patients and normal controls, the functional studies indicate that IRS protein activation status is implicated in the biology of CML cells. NT157 pharmacological IRS1/2 inhibition (i) reduces colony formation in primary CML, but not in normal cells, (ii) decreases cell viability and proliferation, and (iii) increases apoptosis of K562 cells in a time and dose-dependent manner. Since IRS2 lentiviral-mediated silencing did not reduce K562 cell viability, IRS1 inhibition may be the main mechanism by which NT157 exerts its effects on BCR-ABL1 positive cells. NT157 IRS1/2-targeting may optimize the anti-CML approaches.