Pharmacological IRS1/2 Inhibition Induces Apoptosis in BCR-ABL1^T315I mutant Cells

Introduction: Chronic myeloid leukemia (CML) is a hematological malignancy associated with the BCR-ABL1 oncoprotein, which results in deregulated kinase activity-driven leukemic phenotype. Most CML cases are successfully treated with tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1, but a significant percentage of patients develop drug resistance and/or progress to blast crisis, a disease stage that is refractory to therapy. Furthermore, recent studies have provided evidence that the CML leukemia stem cell is resistant to imatinib treatment. In this sense, the identification of other crucial proteins that cooperate with BCR-ABL1 signaling pathways may represent secondary therapeutic targets optimize treatment strategies. Increased IRS1 mRNA expression has been previously identified as positively correlated with worse overall survival in BCR-ABL1-positive adult acute lymphoblastic leukemia. In K562 cells, IRS1 has been identified as a binding partner of BCR-ABL1 and is capable of activating the 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 have previously shown that NT157 treatment reduces colony formation of primary CML cells but not in normal cells, decreases cell viability and proliferation of K562 cells, and increases apoptosis of K562 cells in a time- and dose-dependent manner (ASH 2015 - Abstract #83876). To further characterize the mechanism of the sensitivity of CML cells to IRS1/2 inhibition, here we performed PCR-based gene expression analyses in CML cells in response to NT157 treatment and investigated the effects of IRS1/2 inhibition on cell proliferation and apoptosis in Ba/F3 cells expressing wild-type or T315I mutant BCR-ABL1.

Materials and Methods: K562 cells treated with vehicle or with the IRS1/2 pharmacological inhibitor NT157 (1.6 µM) for 48 hours were submitted to PCR array analysis using the PI3K-AKT Signaling Pathway and CancerPathwayFinder RT2 Profiler PCR Array kit (#PAHS-033A and #PAHS-058A, respectively; SA Biosciences, Frederick, MD, USA). Each cDNA sample was processed in a 96-well plate containing 156 signaling pathway-related genes and 5 endogenous control genes. Ba/F3 parental, BCR-ABL1^WT and BCR-ABL1^T315I cells were subjected to IRS1/2 pharmacological inhibition using NT157 (0.2, 0.4, 0.8, 1.6 and 3.2 µM) for 24, 48 and 72 hours and were evaluated for cell viability (MTT assay), apoptosis (annexin V/PI), and protein expression/activation (Western blot). Statistical analyses were performed by Student's t-test or Mann-Whitney test, as appropriate.

Results: Sixteen genes were found to be differentially expressed (fold change ≥ 2); 5 upregulated genes (FOXO3A, CDKN1A, FOS, JUN, VEGF) and 1 downregulated gene (E2F1) were chosen for validation in a larger number of experiments. Notably, NT157 treatment also resulted in significant dose- and time-dependent decrease in viability in BCR-ABL1-expressing cells regardless of BCR-ABL1 mutation status, compared to parental Ba/F3 cells. NT157 treatment (1.6 µM) for 48 hours reduced cell viability of Ba/F3 BCR-ABL1^WT and BCR-ABL1^T315I cells to 64% and 76%, respectively (p<0.05). Identical treatment with NT157 also significantly induced apoptosis as noted by the percentage of cells positive for annexinV/PI staining compared to vehicle in Ba/F3 BCR-ABL1^WT and BCR-ABL1^T315I cells (84 vs. 13% and 92 vs. 22%, respectively; p<0.05). Similar results were observed after 24 and 72 hours of NT157 treatment for both cell lines. More importantly, NT157 treatment reduced phosphorylation of STAT3 at both serine (S)727 and tyrosine (Y)705 in Ba/F3 BCR-ABL1^T315I in the absence of IL3.

Conclusion: Pharmacological inhibition of IRS1/2 decreases cell viability, increases apoptosis, and reduces STAT3 activation in cells expressing wild-type or T315I-mutant BCR-ABL1 in a time- and dose-dependent manner. These findings suggest that targeting of IRS1/2 alone or in combination with ABL1 TKIs may offer further improved treatment strategies and outcomes for maximizing control of disease resistance and persistence in patients with CML.