Selectivity and therapeutic inhibition of kinases: to be or not to be?

Protein kinases, which serve critical functions in signaling pathways in all cells, are popular therapeutic targets. At present, eight kinase inhibitors have been approved in the United States, each of which shows nanomolar potency. Although the initial goal was to generate inhibitors with a high degree of selectivity, recent experience has revealed that many of these approved compounds target more than one kinase. Surprisingly, this promiscuity is less problematic than one would have imagined; indeed, it opens new therapeutic opportunities. In this Perspective, we discuss the present status of Janus kinase inhibitors-a new class of immunosuppressive drugs-and the advantages and disadvantages of selectively inhibiting this class of kinase.

JAK2 inhibitors for myeloproliferative neoplasms: what is next?

Since its approval in 2011, the Janus kinase 1/2 (JAK1/2) inhibitor ruxolitinib has evolved to become the centerpiece of therapy for myelofibrosis (MF), and its use in patients with hydroxyurea resistant or intolerant polycythemia vera (PV) is steadily increasing. Several other JAK2 inhibitors have entered clinical testing, but none have been approved and many have been discontinued. Importantly, the activity of these agents is not restricted to patients with JAK2 V617F or exon 12 mutations. Although JAK2 inhibitors provide substantial clinical benefit, their disease-modifying activity is limited, and rational combinations with other targeted agents are needed, particularly in MF, in which survival is short. Many such combinations are being explored, as are other novel agents, some of which could successfully be combined with JAK2 inhibitors in the future. In addition, new JAK2 inhibitors with the potential for less myelosuppression continue to be investigated. Given the proven safety and efficacy of ruxolitinib, it is likely that ruxolitinib-based combinations will be a major way forward in drug development for MF. If approved, less myelosuppressive JAK2 inhibitors such as pacritinib or NS-018 could prove to be very useful additions to the therapeutic armamentarium in MF. In PV, inhibitors of histone deacetylases and human double minute 2 have activity, but their role, if any, in the future treatment algorithm is uncertain, given the availability of ruxolitinib and renewed interest in interferons. Ruxolitinib is in late-phase clinical trials in essential thrombocythemia, in which it could fill an important void for patients with troublesome symptoms.

Ruxolitinib reduces JAK2 p.V617F allele burden in patients with polycythemia vera enrolled in the RESPONSE study.

In patients with polycythemia vera (PV), an elevated JAK2 p.V617F allele burden is associated with indicators of more severe disease (e.g., leukocytosis, splenomegaly, and increased thrombosis risk); however, correlations between allele burden reductions and clinical benefit in patients with PV have not been extensively evaluated in a randomized trial. This exploratory analysis from the multicenter, open-label, phase 3 Randomized Study of Efficacy and Safety in Polycythemia Vera With JAK Inhibitor INCB018424 Versus Best Supportive Care trial evaluated the long-term effect of ruxolitinib treatment on JAK2 p.V617F allele burden in patients with PV. Evaluable JAK2 p.V617F-positive patients randomized to ruxolitinib (n = 107) or best available therapy (BAT) who crossed over to ruxolitinib at week 32 (n = 97) had consistent JAK2 p.V617F allele burden reductions throughout the study. At all time points measured (up to weeks 208 [ruxolitinib-randomized] and 176 [ruxolitinib crossover]), mean changes from baseline over time in JAK2 p.V617F allele burden ranged from -12.2 to -40.0% (ruxolitinib-randomized) and -6.3 to -17.8% (ruxolitinib crossover). Complete or partial molecular response was observed in 3 patients (ruxolitinib-randomized, n = 2; ruxolitinib crossover, n = 1) and 54 patients (ruxolitinib-randomized, n = 33; ruxolitinib crossover, n = 20; BAT, n = 1), respectively. Among patients treated with interferon as BAT (n = 13), the mean maximal reduction in allele burden from baseline was 25.6% after crossover to ruxolitinib versus 6.6% before crossover. Collectively, the data from this exploratory analysis suggest that ruxolitinib treatment for up to 4 years provides progressive reductions in JAK2 p.V617F allele burden in patients with PV who are resistant to or intolerant of hydroxyurea. The relationship between allele burden changes and clinical outcomes in patients with PV remains unclear.

Masked polycythaemia vera: presenting features, response to treatment and clinical outcomes.

Masked polycythaemia vera (PV) has been proposed as a new entity with poorer outcome than overt PV. In this study, the initial clinical and laboratory characteristics, response to treatment and outcome of masked and overt PV were compared using red cell mass and haemoglobin or haematocrit levels for the distinction between both entities. Sixty-eight of 151 PV patients (45%) were classified as masked PV according to World Health Organisation diagnostic criteria, whereas 16 (11%) were classified as masked PV using the British Committee for Standards in Haematology (BCSH). In comparison with overt PV, a higher platelet count and a lower JAK2V617F allele burden at diagnosis were observed in masked PV. Patients with masked PV needed lower phlebotomies and responded faster to hydroxcarbamide than those with overt PV. Complete haematological response was more frequently achieved in masked than in overt PV (79% vs. 58%, P = 0.001). There were no significant differences in the duration of haematological response, the rate of resistance or intolerance to hydroxycarbamide and the probability of molecular response according to type of PV (masked vs. overt). Overall survival, rate of thrombosis and major bleeding, and probability of transformation was superimposable among patients with masked and overt PV.

ruxolitinib (JAKAVI°) and polycythaemia vera Inconclusive evaluation.

Patients with polycythaemia vera, a myeloproliferative syndrome, are at increased risk of thrombotic events. Marrow fibrosis and transformation to acute leukaemia can also occur after several years. Treatment is based on phlebotomy and aspirin at low (antiplatelet) doses, sometimes combined with hydroxycarbamide, a cytotoxic drug. Various other drugs are available if hydroxycarbamide fails or is poorly tolerated, but there is no consensus treatment. Ruxolitinib inhibits Janus tyrosine kinases, which are involved, among other roles, in haematopoiesis. Ruxolitinib has been authorised in the European Union for patients with polycythaemia vera in whom hydroxycarbamide has failed or is poorly tolerated. Clinical evaluation of ruxolitinib in this setting is based on a randomised, unblinded trial versus treatment chosen by the investigators, in 222 patients treated for 32 weeks. Hydroxycarbamide was chosen in 59% of cases, even though the patients had unacceptable adverse effects or an inadequate response to the drug. The efficacy of the investigators' other treatment choices was uncertain. Phlebotomy was less frequent in the ruxofitinib group than in the control group. The adverse effects of ruxotitinib in this situation are poorly documented, due to inadequate long-term assessment. In the short term, ruxolitinib causes anaemia and thrombocytopenia, as well as bleeding, potentially severe infections, headache, sensory disturbances, and weight gain. It is also likely to share the serious adverse effects of other immunosuppressants. Ruxolitinib is mainly metabolised by cytochrome P450 isoenzymes CYP3A4 and CYP2C9, creating a risk of multiple drug interactions. Ruxolitinib showed embryofetal toxicity in animal studies. Virtually no data are available in pregnant women. In practice, available data on the harm-benefit balance of ruxolitinib fail to show that this drug represents a tangible advance for patients with polycythaemia vera, as compared with other drugs used when hydroxycarbamide is unsuitable.

Evaluation of WHO criteria for diagnosis of polycythemia vera: a prospective analysis.

We prospectively evaluated the accuracy of the 2007 World Health Organization (WHO) criteria for diagnosing polycythemia vera (PV), especially in "early-stage" patients. A total of 28 of 30 patients were diagnosed as PV owing to an elevated Cr-51 red cell mass (RCM), JAK2 positivity, and at least 1 minor criterion. A total of 18 PV patients did not meet the WHO criterion for an increased hemoglobin value and 8 did not meet the WHO criterion for an increased hematocrit value. Bone marrow morphology was very valuable for diagnosis. Low serum erythropoietin (EPO) values were specific for PV, but normal EPO values were found at presentation (20%). We recommend revision of the WHO criteria, especially to distinguish early-stage PV from essential thrombocythemia. Major criteria remain JAK2 positivity and increased red cell volume, but Cr-51 RCM is mandatory for patients who do not meet the defined elevated hemoglobin or hematocrit value (>18.5 g/dL and 60% in men and >16.5 g/dL and 56% in women, respectively). Minor criteria remain bone marrow histology or a low serum EPO value. For patients with a normal EPO value, marrow examination is mandatory for diagnostic confirmation. Because the therapies for myeloproliferative disorders differ, our data have major clinical implications.

JAK2 p.V617F detection and allele burden measurement in peripheral blood and bone marrow aspirates in patients with myeloproliferative neoplasms.

Detection of the JAK2 p.V617F mutation and measurement of its allele burden can be performed using both peripheral blood (PB) and bone marrow (BM) samples from patients with myeloproliferative neoplasms (MPNs). However, the diagnostic accuracy of detecting the JAK2 p.V617F mutation and quantifying its allele burden in PB and BM samples has not been systematically compared. We retrospectively analyzed 388 patients with MPN who had been tested for JAK2 p.V617F allele burden using both PB and BM samples within 3 months of each other. The sensitivity and specificity of detecting JAK2 p.V617F in PB when compared with BM were both 100%. Furthermore, the JAK2 p.V617F allele burden measured in PB and BM were equivalent by linear regression analysis (R(2) = 0.991; P < .0001). We therefore conclude that PB is a reliable source for testing for the JAK2 p.V617F mutation and quantifying its allele burden in patients with MPN.

Current and future treatment options for polycythemia vera.

Patients with polycythemia vera (PV), a myeloproliferative neoplasm characterized by an elevated red blood cell mass, are at high risk of vascular and thrombotic complications and have reduced quality of life due to a substantial symptom burden that includes pruritus, fatigue, constitutional symptoms, microvascular disturbances, and bleeding. Conventional therapeutic options aim at reducing vascular and thrombotic risk, with low-dose aspirin and phlebotomy as first-line recommendations for patients at low risk of thrombotic events and cytoreductive therapy (usually hydroxyurea or interferon alpha) recommended for high-risk patients. However, long-term effective and well-tolerated treatments are still lacking. The discovery of mutations in Janus kinase 2 (JAK2) as the underlying molecular basis of PV has led to the development of several targeted therapies, including JAK inhibitors, and results from the first phase 3 clinical trial with a JAK inhibitor in PV are now available. Here, we review the current treatment landscape in PV, as well as therapies currently in development.

Open-label study of oral CEP-701 (lestaurtinib) in patients with polycythaemia vera or essential thrombocythaemia with JAK2-V617F mutation.

JAK2-V617F is central to the pathogenesis of myeloproliferative neoplasms. We examined whether lestaurtinib decreased JAK2-V617F allele burden and evaluated its clinical benefits and tolerability in patients with polycythaemia vera (PV) and essential thrombocythaemia (ET). This phase 2, open-label, multicentre study was designed to detect ≥15% reduction in JAK2-V617F allele burden in 15% of patients. Eligible patients received lestaurtinib 80 mg twice daily for 18 weeks and could participate in a 1-year extension phase of treatment. Of 39 enrolled patients, 27 (69%) had PV; 12 (31%) had ET. While the pre-specified responder rate of 15% was not met, lestaurtinib modestly reduced JAK2-V617F allele burden and reduced spleen size in a subset of patients. Of 37 patients in the full efficacy analysis, 5 (14%) responded clinically. Every patient had ≥1 adverse event, most commonly gastrointestinal (95%). Fifteen patients (38%) experienced serious adverse events; 23 (59%) withdrew due to adverse events. This is the first reported study of JAK2-inhibitor treatment in patients with PV/ET and highlights both the need for further studies to assess the role of JAK2 inhibition in treatment of PV/ET and the use of JAK2-V617F as a biomarker for response. This trial was registered at www.clinicaltrials.gov as NCT00586651.

Discriminating between essential thrombocythemia and masked polycythemia vera in JAK2 mutated patients.

In patients not meeting the required hematocrit (HCT) or hemoglobin (Hb) thresholds according to BCSH and the WHO diagnostic criteria, the diagnosis of masked polycythemia vera (mPV) has been proposed. A comparison of HCT or Hb values with the expression of JAK2V617F, JAK2 exon 12, and CALR mutations in strictly WHO-defined 257 overt PV and 140 mPV (59 mPV according to BCSH) and 397 patients with essential thrombocythemia (ET) was performed. Hb and HCT thresholds of mPV patients were significantly higher than JAK2V617F ET (P < 0.0001). The best cut-off for Hb to discriminate JAK2-mutated ET from PV was 16.5 g/dL for males and 16.0 g/dL for females. For HCT, this was 49% in males and 48% in females. The proportion of patients correctly classified as ET or PV when regarding Hb or HCT levels was 95% in males and 93% in females and 94% in both males and females, respectively.

JAK2V617F monitoring in polycythemia vera and essential thrombocythemia: clinical usefulness for predicting myelofibrotic transformation and thrombotic events.

The JAK2V617F allele burden has been identified as a risk factor for vascular events and myelofibrotic transformation in polycythemia vera (PV) and essential thrombocythemia (ET). However, all previous studies have evaluated a single time point JAK2V617F measurement. Therefore, the frequency and the clinical significance of changes in the JAK2V617F mutant load occurring during the disease evolution remain unknown. In the present study, JAK2V617F monitoring was performed during the follow-up of 347 patients (PV = 163, ET = 184). According to their JAK2V617F evolutionary patterns, patients were stratified as stable < 50% (n = 261), stable ≥50% (n = 52), progressive increase (n = 24) and unexplained decrease (n = 10). After a 2,453 person-years follow-up, a total of 59 thrombotic events, 16 major hemorrhages, and 27 cases of myelofibrotic transformations were registered. At multivariate analyses, patients with a persistently high (≥50%) or unsteady JAK2V617F load during follow-up had an increased risk of myelofibrotic transformation (Incidence rate ratio [IRR]: 20.7, 95% CI: 6.5-65.4; P < 0.001) and a trend for a higher incidence of thrombosis (IRR: 1.7, 1-3.3; P = 0.05) than patients with a stable allele burden below 50%. In conclusion, JAK2V617F monitoring could be useful in patients with PV and ET for predicting disease's complications, especially myelofibrotic transformation.

Inhibition of related JAK/STAT pathways with molecular targeted drugs shows strong synergy with ruxolitinib in chronic myeloproliferative neoplasm.

This study aimed to assess the antitumour effects, molecular mechanisms of action, and potential synergy of ruxolitinib with sorafenib, KNK437, dasatinib, and perifosine, in Philadelphia-negative chronic myeloproliferative neoplasms (MPN). Cytotoxic and cytostatic effects of the different compounds were determined in the JAK2 V617F-positive cell lines, HEL and Ba/F3 (JAK2V617F EPOR) , and in primary mononuclear and bone marrow CD34-positive cells from 19 MPN patients. Ruxolitinib [50% inhibitory concentration (IC50 )(PV)  = 15 nmol/l], as well as sorafenib (IC50 PV=8µmol/l), KNK437 (IC50 PV=100µmol/l ), and perifosine (IC50 PV=15µmol/l ), were able to inhibit proliferation in cell line models and in primary cells from MPN patients. Dasatinib, KNK437, and sorafenib showed a strong synergistic effect in combination with ruxolitinib [combination index (CI)(PV)  < 0·3]. Western blot confirmed that ruxolitinib blocked ERK, and consequently STAT5 activation, sorafenib inhibited ERK, P38 and STAT5, dasatinib blocked SRC and STAT5, and KNK437 decreased the stability of the JAK2 protein, reducing its expression. Inhibiting JAK2-related proliferative pathways has the potential to inhibit cell proliferation in MPNs. Furthermore, the combination of ruxolitinib with inhibitors that target these pathways has a strong synergistic effect, which may be due to decreased activation of the common effector, STAT5.

New insights into the structure and function of the pseudokinase domain in JAK2.

JAK (Janus kinase) 2 plays a critical role in signal transduction through several cytokine receptors. JAKs contain a typical tyrosine kinase domain preceded by a pseudokinase [JH2 (JAK homology 2)] domain which has been considered to be catalytically inactive. Identification of activating mutations in the JH2 domain of JAK2 as the major cause for polycythaemia vera and other MPNs (myeloproliferative neoplasms) demonstrate the critical regulatory function for this domain, but the underlying mechanisms have remained elusive. We have performed biochemical and functional analysis on the JH2 domain of JAK2. The results indicate that JH2 functions as an active protein kinase and phosphorylates two residues in JAK2 (Ser523 and Tyr570) that have been shown previously to be negative regulatory sites for JAK2 activity. The crystal structure of the JAK2 JH2 domain provides an explanation for the functional findings and shows that JH2 adopts a prototypical kinase fold, but binds MgATP through a non-canonical mode. The structure of the most prevalent pathogenic JH2 mutation V617F shows a high level of similarity to wild-type JH2. The most notable structural deviation is observed in the N-lobe αC-helix. The structural and biochemical data together with MD (molecular dynamics) simulations show that the V617F mutation rigidifies the αC-helix, which results in hyperactivation of the JH1 domain through an as yet unidentified mechanism. These results provide structural and functional insights into the normal and pathogenic function of the JH2 domain of JAK2.

JAK2V617F mutation and hydroxyurea treatment as determinants of immature platelet parameters in essential thrombocythemia and polycythemia vera patients.

Immature platelets (IPFs), which are hemostatically more active than mature platelets, have been found elevated in essential thrombocythemia and polycythemia vera, 2 myeloproliferative neoplasms (MPN) characterized by an increased risk of thrombosis. It is not known whether the IPF levels are influenced by pathogenetic factors, including JAK2V617F mutational status, or by treatment regimen. To address this point, in 46 essential thrombocythemia and 38 polycythemia vera consecutive patients, we measured IPF and correlated the results to JAK2V617F mutation and myelosuppressive treatment with hydroxyurea. This analysis provides 2 new elements regarding IPF and MPN. The first finding is that the JAK2V617F mutation is linked to the quantity of IPF in patients with MPN, which might contribute to the prothrombotic phenotype in these patients. The second finding is that IPF is susceptible to myelosuppressive treatment, which may additionally explain the favorable effect of hydroxyurea therapy on MPN outcome as well as the associated thrombotic risk.

Increased basal intracellular signaling patterns do not correlate with JAK2 genotype in human myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are associated with recurrent activating mutations of signaling proteins such as Janus kinase 2 (JAK2). However, the actual downstream signaling events and how these alter myeloid homeostasis are poorly understood. We developed an assay to measure basal levels of phosphorylated signaling intermediates by flow cytometry during myeloid differentiation in MPN patients. Our study provides the first systematic demonstration of specific signaling events and their comparison with disease phenotype and JAK2 mutation status. We demonstrate increased basal signaling in MPN patients, which occurs in both early and later stages of myeloid differentiation. In addition, the pattern of signaling is not correlated with JAK2 mutation status and signaling intensity is poorly correlated with mutant JAK2 allele burden. In contrast, signaling differences are detected between different MPN disease phenotypes. Finally, we demonstrate that signaling can be inhibited by a JAK2-selective small molecule, but that this inhibition is not JAK2 V617F specific, because MPN patients with mutant JAK2, wild-type JAK2, and control patients were inhibited to a similar degree. Our data suggest that, in addition to JAK2 mutations, other factors contribute significantly to the MPN phenotype, results that are relevant to both the pathogenesis and therapy of MPN.

Aberrant expression of signaling proteins in essential thrombocythemia.

Dysregulated expression of signaling proteins may contribute to the pathophysiology of essential thrombocythemia (ET). This study aimed to characterize protein expression in ET and to correlate the dysregulated proteins with phenotypes and prognosis of ET patients. The expression of 128 proteins in peripheral blood neutrophils from 74 ET patients was assessed and compared with those from 29 healthy subjects and 35 polycythemia vera (PV) patients using protein pathway array. Fifteen proteins were differentially expressed between ET patients and normal controls. These dysregulated proteins were involved in the signaling pathways related with apoptosis and inflammation. Our results showed a significant overlap in protein expression between ET patients with JAK2V617F mutation and PV patients. In addition, nine proteins were associated with JAK2V617F mutation status in ET patients. Furthermore, estrogen receptor beta (ERß) and Stat3 were independent risk factors for subsequent thrombosis during follow-up on multivariable analysis. Our study shows a broad dysregulation of signaling protein in ET patients, suggesting their roles in ET pathogenesis. The expression levels of ERß and Stat3 could be promising predictors of subsequent thrombosis in ET patients.

Effects of imatinib mesylate in patients with polycythemia vera: results of a phase II study.

The antiproliferative effects of the tyrosine kinase inhibitor imatinib mesylate were reported in single cases with polycythemia vera (PV). We, therefore, conducted a clinical phase II study to assess the rate and quality of response in patients with PV. Thirty-one patients, with a median age of 64 years (range, 45-84 years), were included. All but one patient were on phlebotomy; 14 (45%) had previously received cytoreductive therapy. Imatinib was started with 400 mg/day. In 26% of patients, dose escalation up to 800 mg was performed. After a median treatment duration of 8.3 months (range, 0.1-62.1 months), the overall response rate was 55%. No complete remission (normalization of all parameters: hematocrit, white blood cell (WBC) count, platelet count, and spleen size determined by ultrasound examination) was reached because the spleen remained enlarged in all patients. Thirteen patients (42%) achieved partial remission (≥25% reduction of at least one of the previously mentioned parameters); in 10 of these, the respective reduction was ≥50%. In four patients (13%) with minor response, the reduction was <25%. No change or progressive disease was seen in 14 patients (45%). The nonresponders had a longer previous disease duration and had received more antecedent cytoreductive therapy (p = 0.009). Compared to baseline characteristics, imatinib induced the reduction of phlebotomies (p = 0.003), of WBC count (p = 0.002), and of platelet count (p = 0.04). Three patients became free from phlebotomies. In six investigated patients, no significant reduction of the JAK2V617F burden was observed despite clinical improvement. The results show that imatinib has moderate cytoreductive effects in PV.

The expression of lysyl-oxidase gene family members in myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are malignant disorders originating from clonal expansion of a single neoplastic stem cell and characteristically show an increase in bone marrow reticulin fibers. Lysyl oxidases (LOXs) are copper-dependent amine oxidases that play a critical role in the biogenesis of connective tissue by crosslinking extracellular matrix proteins, collagen and elastin. Expression of LOX gene family members is increased in disorders associated with increased fibrosis. To evaluate involvement of LOX gene family in various MPNs. In-situ hybridization was used to detect Lysyl-Oxidase family members in bone marrow biopsies from patients with different MPNs. We compared normal bone marrows and those from patients with polycythemia vera, essential thrombocythemia, chronic myeloid leukemia, and primary myelofibrosis (PMF). Serum levels of lysyl-oxidase from patients with PMF and healthy controls were also examined. LOX gene family was not detected in normal bone marrows. All members of the LOX gene family were over expressed in PMF. In other MPNs a differential pattern of expression was observed. Differences in gene expression were statistically significant (P < 0.010). The medianserum LOX levels in normal controls was 28.4 ± 2.5 ng\ml and 44.6 ± 9.44 ng\ml in PMF (P = 0.02). The varying pattern of expression of LOX genes may reflect differences in the pathophysiology of bone marrow fibrosis in these MPNs. These observations could be used as the basis for future targeted therapy directed against bone marrow fibrosis.