Expression of JAK2-V617F Kinase in Myeloid Progenitors and Proerythroblasts Induces Differential Patterns of Hypersensitivity in Activation of Key Signaling Nodes upon Incubation with Low-Dose, Physiologic EPO Concentrations

Introduction:

Chronic myeloproliferative neoplasms (MPNs), including Polycythemia vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are a spectrum of clonal hematological disorders. An acquired somatic mutation of the JAK2-gene (JAK2^V617F) drives clonal expansion of hematopoietic progenitor cells in 90-95% in cases of PV and in approximately 40-50% in ET and PMF patients. This mutation induces an aberrant cellular signaling affecting the activation of several downstream protein cascades of cytokine receptors and substrates of the JAK2 protein. This constitutive activation leads to deregulated proliferation and differentiation of one or several myeloid lineages. Studies have shown that progenitor cells derived from JAK2^V617F-positive patients are characterized by hypersensitivity to cytokines, e.g. erythropoietin (EPO), leading to an increased proliferation and disturbed differentiation under low –dose cytokine treatment.

Methods:

Murine 32D progenitor cells and I-11 proerythroblasts were transduced with retroviral vectors expressing cDNAs of the erythropoietin receptor and JAK2^WT/JAK2^V617F. Stably transduced cell lines were treated with increasing concentrations of EPO. To study the kinetics within the EPO signaling network, cell lines were starved for 4h and then treated with increasing doses of EPO (1 – 10 IU EPO for 32D, 0.5 – 3 IU EPO for I-11) for 1h (32D cells) and for 30min (I-11 cells). Phosphorylation of STAT3/STAT5, JAK2, Erk1/2, Akt and PLCg1, respectively were detected by immunoblot analysis. Densitometric analysis of immunoblot-signals (phospho-signals adjusted to total protein signals) was performed to quantitatively determine differences in phosphorylation of the main molecular pathways of EPO signaling.

Results:

Treatment with low physiologic doses of EPO resulted in an enhanced cell proliferation in JAK2^V617F expressing cells compared to JAK2^WT. However, this effect leveled off upon EPO concentrations >0.75 IU/ml. To investigate the molecular mechanisms of this hypersensitive status, we quantitatively monitored dose- and time-dependent phosphorylation of signaling proteins, which play a major role in the EPO signaling network, including STAT3/STAT5, Erk1/2, Akt and PLCg1. In unstimulated JAK2^V617F mutated 32D and I-11 cells, we identified constitutive activation of these key signaling regulators. Moreover, we could demonstrate that in JAK2^V617F expressing cells EPO dependent activation of these key signaling molecules is significantly more sensitive to low EPO concentrations as compared to the situation in JAK2^WT expressing cells. Thus, we detected a higher peak of phosphorylation/activation of STAT3/STAT4, Erk1/2, Akt and PLCg1 in low-dose treated JAK2^V617F cells. In addition, in JAK2^V617F positive cells, three patterns of signaling kinetics were observed: 1. Left shift of activation curve and higher maximum of activation which is overcome by high EPO concentrations (e.g. phospho-JAK2); 2. Left shift of activation curve and higher maximum of activation which cannot be overcome by high EPO concentrations (e.g. phospho-STAT5); 3. Pattern exhibiting minor differences only (e.g. phospho-ERK1/2). We hypothesize that this is due to differential activation of feedback and feedforward loops. Quantitative and qualitative modelling is currently being performed to identify the molecular mechanisms involved.

Along this line, we identified the docking protein Grb2-associated-binding protein1 (Gab1) as a member of EPO-dependent proteins. Gab1 plays a major role in co-activation of MAPK- and PI3K-pathway. Thus, for the first time we demonstrate constitutive activation of Gab1 in JAK2^V617F mutated cells.

Conclusions:

We here demonstrate that hypersensitivity in proliferation of JAK2^V617F mutated progenitor cells and proerythroblasts is molecularly corroborated by differential sensitivity of the pro-proliferative signaling network. We identified at least four different signaling pathways, which show higher sensitivity for activation in JAK2^V617F mutated cells compared wild type cells. Hypersensitivity to low-dose EPO treatment on a molecular level may explain some of the biological features observed in JAK2^V617F positive MPNs and may offer novel targets for therapeutic intervention.