The JAK2 blocker TG101209 is a potent inhibitor of clonogenic progenitor cell growth in patients with chronic myeloid leukaemia (2017)

Janus kinase (JAK) 2 is a non-receptor tyrosine kinase (TK) that is essential for signalling through a variety of cytokine receptors. In normal haematopoiesis JAK2 is activated when haematopoietic growth factors bind to their receptors. Activated JAK2 opens the path to downstream signalling via STAT proteins, inducing genes that are essential for haematopoietic cell growth (Ward et al, 2000). Dysregulated JAK-STAT signalling leading to uncontrolled growth of neoplastic cells is a hallmark of malignant haematopoiesis. BCR-ABL1 negative myeloproliferative neoplasms (MPNs) harbouring JAK2 activation mutations represent the prototype of haematopoietic malignancies with constitutively active JAK2/STAT5 signalling (James et al, 2005). These findings have led to the development of small-molecule inhibitors that target JAK2, and the JAK1/JAK2 inhibitor ruxolitinib is already approved for treatment of primary myelofibrosis and polycythaemia vera. JAK2 has also been shown to be involved in chronic myeloid leukaemia (CML) leukaemogenesis, suggesting JAK2 as a potential therapeutic target in CML (Samanta et al, 2006). These findings prompted us to investigate the effect of the JAK2-targeting- inhibitor TG101209 on in-vitro clonogenic growth of peripheral blood progenitor cells from 36 untreated patients with newly diagnosed CML in chronic phase. Untreated patients with BCR-ABL1 negative MPN harbouring the JAK2 V617F mutation (n = 14) and patients without haematological malignancy (n = 9) were included as control cohorts. The study was approved by the ethics committee of the Medical University of Vienna, Austria (EK 2006/2014). Peripheral blood mononuclear cells (50–100 × 106/l) were cultured in 0·8% methylcellulose (Sigma, Saint Louis, MO, USA), 30% fetal calf serum (Linaris, Wertheim-Bettingen, Germany), 10% bovine serum albumin (Stem Cell Technologies Inc., Vancouver, Canada), α-thioglycerol (10−4 mol/l) (Fluka Chemie AG, Buchs, Switzerland) and Iscove's modified Dulbecco's medium (Gibco-Invitrogen, Paisley, UK). Colony growth was stimulated by adding 5 ng/ml recombinant human interleukin-3 (rh-IL3) (R&D Systems, Minneapolis, MN, USA), 10 ng/ml rh granulocyte-macrophage colony-stimulating factor (R&D Systems, Minneapolis), and 2 IU/ml rh erythropoietin (Roche, Mannheim, Germany) to the cultures. All cultures were plated in duplicate. The JAK2 -inhibitor TG101209 (Symansis, Temecula, CA, USA) was added to cultures at concentrations of 500 nM, 100 nM or 10 nM. The parallel baseline assay was supplemented with dimethylsulfoxide only. After 14 days culture at 37°C in 5% CO2 and full humidity, the cultures were examined under an inverted microscope. Aggregates with more than 40 translucent, compact or dispersed cells were counted as colony forming units-granulocyte/macrophage (CFU-GM). Aggregates of at least 50 haemoglobinised cells, easily recognizable by their red colour, were counted as burst forming units-erythroid (BFU-E). Pluripotent progenitors (CFU-GEMM) were identified by their heterogeneous composition of translucent and haemoglobinized cells. Results of inhibition experiments were expressed as per cent inhibition from baseline value. The one sample t-test (Ho: mean change = 0) was applied to assess changes to baseline at different concentrations of TG101209 within each patient group. Differences between the 3 groups were tested with the Kruskal–Wallis test. In case of significance in the global test single group comparisons were performed using the Wilcoxon rank sum test. Spearman correlation was used to assess correlations between the growth inhibitory effects of TG101209 and clinical parameters. TG101209 exerted a dose-dependent inhibitory effect on all types of colonies in each patient cohort (Fig 1). At the highest concentration (500 nM) the degree of inhibition from baseline level was similar for all three groups (change to baseline: P < 0·0001, each). At a concentration of 100 nM, the degree of inhibition remained highly significant for CML and MPN groups (change to baseline: P < 0·0001, each), and was still significant for CFU-GM (P = 0·0012) and BFU-E (P = 0·0007) in nonmalignant samples. Furthermore, at this dose-level a significant difference between the CML and the nonmalignant group was observed in terms of inhibition of BFU- E and GEMM (P < 0·05, each). At the 10 nM concentration, suppression of colony growth from baseline was still highly significant in CML samples (P < 0·0001 for GM and BFU-E; P = 0·0002 for GEMM), whereas it became less significant in JAK2-mutated MPN samples (P < 0·05 for all types of colonies), and was no longer significant in nonmalignant samples. No significant correlation was found between in-vitro effects of TG101209 and pretreatment values of white blood cells, platelets, haemoglobin and basophils, as well as Hasford and Sokal score in patients with CML (data not shown). JAK2-inhibitors, used at concentrations ranging from 100 nM to 12·500 nM, have previously been shown to inhibit in-vitro growth of BCR-ABL1 positive cell lines as well as primary CML cells (Sun et al, 2001; Samanta et al, 2006; Wang et al, 2007; Hiwase et al, 2010; Hantschel et al, 2012; Chen et al, 2013; Gallipoli et al, 2014). The conclusion of most of these studies was that JAK2-inhibitors mainly augmented the growth suppressing effect of BCR-ABL1 TK-inhibitors, rather than provoking a major inhibitory effect per se. This is the first study to demonstrate potent single agent activity of the JAK2 blocker TG101209 in a series of newly diagnosed CML patients. Significant inhibition of clonogenic colonies was achieved at inhibitor concentrations far below those used in the majority of former studies. Likely explanations for these differences include different types of study materials and culture methods. In fact, most of the reported studies had used artificial cell lines and/or primary cells from patients with advanced and/or TK-inhibitor resistant disease and inhibition experiments had been performed either in suspension cultures only or in colony assays after pre-incubation with inhibitors in suspension culture. The present study used exclusively primary cells from patients with chronic phase CML, and the JAK2 -inhibitor was present in the semisolid culture medium throughout the entire assay period. STAT5 signalling plays a central role in the pathogenesis of CML. However, unlike in BCR-ABL1 negative MPNs, the role of JAK2 in activating STAT5 in CML has been challenged by demonstrating that BCR-ABL1 directly phosphorylates STAT5 (Hantschel et al, 2012). Nevertheless, there is accumulating evidence for a growth-promoting role of JAK2 in CML “offside” endogenous activation of STAT5. Postulated mechanisms include direct interaction with BCR-ABL1, inhibition of the tumour suppressor phosphatase protein 2A, as well as extrinsic cytokine-mediated survival signalling (Samanta et al, 2006; Wang et al, 2007; Hiwase et al, 2010; Chen et al, 2013; Neviani et al, 2013). Regardless of the mechanisms involved, our data add substantial evidence for a growth regulatory role of JAK2 in chronic phase CML. P.V. received research support from Novartis and Ariad and honoraria form Novartis, Pfizer and Ariad. S.D., E.J., E.P., G.G., S.H. and I.S. declare no conflict of interest. This study was supported by Austrian Science Fund, grant SFB F4704, to PV. S.D. collected and analysed the data and wrote the paper; E.J. performed the colony assays; E.P. performed the statistical analyses; G.G., S.H. and P.V. contributed clinical data and vital logistic support; P.V. helped to edit the manuscript; I.S. designed and organized the research and edited the manuscript.