IRS2 Inhibition Reduces Cell Proliferation and Has Cumulative Effects with Ruxolitinib in JAK2 V617F Cells

Background: Insulin receptor substrate 2 (IRS2) mediates mitogenic and antiapoptotic signaling from IR, IGF1R, EPOR and TPOR. Studies performed in non-hematological lineages have shown that IRS2 associates with proteins involved in the JAK/STAT, PI3K/Akt/mTOR, and Ras/Raf/MAPK pathways, known to be involved in cell proliferation and survival. Primary Myelofibrosis (PMF), Polycythemia Vera (PV), and Essential Thrombocythemia (ET) are chronic myeloproliferative neoplasms (MPN) frequently associated with the JAK2 V617F mutation, and with hyperactivation of the JAK/STAT, PI3K/Akt/mTOR and Ras/Raf/MAPK pathways. Ruxolitinib was designed to target JAK2, but the lack of complete clinical response in most patients indicates the need for identifying other signaling pathways that may cooperate with JAK2. We have previously reported that IRS2 constitutively associates with JAK2 in HEL cells (JAK2 V617F), though not in U937 and NB4 lineages (JAK2 wild type). IRS2 mRNA expression in CD34⁺ cells was significantly higher in ET patients compared to healthy donors, and in patients harboring the JAK2 V617F mutation versusJAK2 wild type, which suggests that IRS2 is involved in cell proliferation in MPN. We herein aim to characterize the effects of IRS2 silencing and to evaluate the synergistic effect of IRS2 silencing and ruxolitinib treatment on cell growth, clonogenicity, apoptosis, and on the JAK/STAT, PI3K/Akt/mTOR and Ras/Raf/MAPK signaling pathways in JAK2 V617F cells treated or not with ruxolitinib.

Methods: 2 x 10⁵ HEL cells were transduced with shIRS2 or shControl lentiviral-mediated delivery and cultured with stable selection using puromycin. On day 21, cells were tested for IRS2 inhibition by qRT-PCR and immunoblotting. IRS2 silenced and control cells were treated with DMSO or with ruxolitinib (100 and 300 nM) and were submitted to: (1) evaluation of cell growth by methythiazoletetrazolium (MTT) and colony forming assays; (2) assessment of apoptosis by Annexin-V/PI and activated caspases 3, 8 and 9 staining by flow cytometry and by immunoblotting; (3) evaluation of proteins involved in JAK/STAT, PI3K/Akt/mTOR and Ras/Raf/MAPK signaling pathways by immunobloting with specific antibodies. Results were analyzed in a total of eight different experiments.

Results: HEL cell growth was significantly inhibited by IRS2 silencing or ruxolitinib treatment compared to DMSO treated cells, and a cumulative effect was observed upon IRS2 silencing and ruxolitinib treated cells, as evaluated by MTT and colony formation assay (all p≤.02). IRS2 silencing significantly induced apoptosis, and an additive effect between IRS2 silencing and ruxolitinib treatment was also observed in HEL cells, as verified by flow cytometry analysis of Annexin-V and caspases 3, 8 and 9 (all p≤.01). IRS2 silencing alone or associated with ruxolitinib treatment reduced STAT3 and STAT5 tyrosine phosphorylation; however, a cumulative effect of both treatments on STAT5 phosphorylation could not be observed, as ruxolitinib alone is a powerful STAT5 inhibitor. While AKT and p70S6K phosphorylation were significantly reduced in IRS2 silenced cells, the same was not observed in cells treated with ruxolitinib alone. IRS2 silencing did not reduce JAK2 tyrosine phosphorylation.

Conclusions: IRS2 is a binding partner downstream of JAK2 V617F and is involved in cell proliferation pathways that are constitutively activated in MPN harboring this mutation. IRS2 silencing augmented the anti-proliferative and proapoptotic effects of ruxolitinib in JAK2 V617F cells. This cumulative effect might be explained by the decreased AKT and p70S6K phosphorylation induced by IRS2 silencing, but not by ruxolitinib alone. In the concept of multi target treatment, IRS2, in association with ruxolitinib, may be a good therapeutic target for patients with MPN harboring JAK2 V617F mutation.